Liver organ model and establishment method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a liver organ model and an establishment method and application thereof. The establishment method comprises the following steps: pre-treating an obtained liver tissue sample to obtain a mixture containing liver tissue-derived cell clusters, and then carrying out gradient digestion treatment, filtration, lysis and centrifugation to obtain bile duct organs; performing induced culture on the bile duct organ to obtain a liver organ model; wherein the gradient digestion method comprises the step of treating the mixture containing the liver tissue-derived cell mass for 5-10 minutes by sequentially using IV type collagenase with the mass concentration of 0.1%, 0.2% and 0.3%. According to the establishment method of the liver organoid model, provided by the invention, the organoid yield is relatively high, and the prepared organoid is clear in boundary and uniform in shape. A fatty liver model constructed by using the aptamer is sensitive to drug response, can screen drugs for treating fatty liver at high throughput, and provides a basis for clinical screening of novel anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a liver organoid model, its establishment method, and its application. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide, with a global prevalence exceeding 25%, posing a significant public health burden. However, drug development for NAFLD has been slow for a long time, mainly due to the lack of preclinical models that can accurately simulate human pathological characteristics and drug responses.

[0003] Current research primarily relies on two types of traditional models: First, traditional two-dimensional (2D) hepatocyte culture models. These models cannot replicate the three-dimensional microenvironment and complex cell-cell interactions of the liver in vivo, leading to significant differences in drug metabolism and pathological responses compared to the real human situation. Second, animal models. These models exhibit clear species differences, differing from humans in key pathways such as drug metabolism and immune responses. This makes it difficult to accurately predict drug efficacy and toxicity in humans based on data obtained from animal experiments, resulting in low success rates and high costs in new drug development.

[0004] In recent years, breakthroughs in liver organoid technology have provided a new approach for constructing in vitro three-dimensional liver models. These models possess self-updating capabilities and can better simulate the structural and functional characteristics of the liver in vivo. However, in NAFLD research applications, this technology still has significant shortcomings in terms of model standardization and stability: First, the induction efficiency of existing models is unstable, and there is a lack of unified standards for free fatty acid treatment concentrations, leading to poor data comparability between different laboratories; second, drug evaluation systems are mostly limited to lipid droplet morphology observation, failing to integrate comprehensive evaluation indicators for key pathological processes such as liver function and inflammatory responses; furthermore, screening systems for traditional Chinese medicine components are still lacking, and traditional decoctions and crude extracts have poor compatibility with organoid culture systems, urgently requiring the establishment of direct screening methods suitable for active ingredients. In addition, in the critical digestion stage of organoid preparation, existing methods are crude and inefficient, easily leading to two consequences: incomplete digestion, resulting in low organoid yield; and over-digestion, directly destroying the integrity of the organoid structure and impairing cell viability. These problems collectively result in a small number, poor quality, and incomplete function of the final organoids, laying an unstable foundation for subsequent high-quality disease modeling.

[0005] In summary, there is an urgent need in this field for a stable and efficient method to construct human liver organoid models, in order to address the shortcomings of existing technologies in constructing liver organoids and provide a reliable research platform for studying the pathological mechanisms of NAFLD. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a liver organoid model, its establishment method and application, to solve the problems of insufficient number, poor quality and incomplete function of organoids in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] This invention provides a method for establishing a liver organoid model, comprising: preprocessing an obtained liver tissue sample to obtain a mixture containing liver tissue-derived cell clusters;

[0009] The mixture containing liver tissue-derived cell clusters was subjected to gradient digestion. After digestion, the undigested tissue was filtered out, the filtrate was collected, lysed, and centrifuged to obtain bile duct organoids.

[0010] The bile duct organoids were induced and cultured to obtain liver organoid models;

[0011] The gradient digestion method involves sequentially digesting the mixture containing liver tissue-derived cell clusters with type IV collagenase at mass concentrations of 0.1%, 0.2%, and 0.3% for 5 to 10 minutes.

[0012] This invention provides a method for establishing a liver organoid model. The method is simple to construct, low in cost, and produces organoids with clear boundaries, uniform morphology, and high yield.

[0013] In some embodiments of the present invention, the gradient digestion method includes the following three stages:

[0014] First stage: 0.1% type IV collagenase, shaken at 37°C for 5 minutes for digestion;

[0015] Second stage: 0.2% type IV collagenase, shaken at 37°C for 5 minutes for digestion;

[0016] Third stage: 0.3% type IV collagenase, shaken at 37°C for 10 minutes for digestion.

[0017] The gradient digestion method provided in this invention can more gently and efficiently separate bile duct organoids from liver tissue, with a high organoid yield. It avoids the problems of existing technologies that use single-concentration and single-time digestion, which may result in incomplete digestion and reduced organoid activity. Furthermore, single-enzyme digestion may directly damage the organoid structure, leading to organoid death or loss of function, ultimately resulting in a low organoid yield.

[0018] In some embodiments of the present invention, the induction culture method includes: mixing the bile duct organoids with matrix gel in a certain proportion, forming solid droplets after the matrix gel solidifies, followed by amplification culture and differentiation culture, and finally forming a liver organoid model.

[0019] In some embodiments of the present invention, bile duct organoids are uniformly dispersed in the matrix gel.

[0020] In some embodiments of the present invention, the culture time for the amplification culture is 7 to 14 days.

[0021] In some embodiments of the present invention, the amplification culture medium is based on DMEM / F12 and supplemented with 2% (v / v) B27 additive, R-spondin 1 at a final concentration of 200 ng / mL, Noggin at a final concentration of 100 ng / mL, epidermal growth factor (EGF) at a final concentration of 50 ng / mL, fibroblast growth factor 10 (FGF10) at a final concentration of 50 ng / mL, and oncostatin M at a final concentration of 10 ng / mL.

[0022] In some embodiments of the present invention, the culture time for the differentiation culture is 10 to 15 days.

[0023] In some embodiments of the present invention, the culture medium for differentiation culture includes: DMEM / F12 as the basal medium, supplemented with 2% (v / v) B27 additive, 1% (v / v) insulin-transferrin-selenium complex (ITS), 30 ng / mL hepatocyte growth factor (HGF), 20 ng / mL oncostatin M, 5 ng / mL fibroblast growth factor 4 (FGF4), 10 ng / mL bone morphogenetic protein 7 (BMP7), 10 μM A83-01, 5 μM DAPT, and 0.5 μM dexamethasone.

[0024] The present invention also provides a liver organoid model constructed by the aforementioned method.

[0025] This invention also provides the application of the aforementioned liver organoid model in constructing liver disease models.

[0026] In some embodiments of the present invention, the liver disease model is a fatty liver disease model.

[0027] The present invention also provides the application of the liver organoid model in drug screening for fatty liver.

[0028] The present invention also provides a method for constructing a fatty liver organoid model, comprising: inducing the liver organoid model with free fatty acids to obtain the fatty liver organoid model.

[0029] In some embodiments of the present invention, the free fatty acids include palmitic acid and oleic acid; in the induction culture medium, the final concentration of palmitic acid is 200 μmol / L to 1000 μmol / L, and the final concentration of oleic acid is 200 μmol / L to 1000 μmol / L. The present invention selects palmitic acid and oleic acid as free fatty acids, and by selecting these two free fatty acids and their concentrations, the physiological lipid environment of the liver can be simulated to achieve precise induction of liver organoids.

[0030] In some specific embodiments of the present invention, the final concentration of palmitic acid in the induction culture medium is 200 μmol / L, and the final concentration of oleic acid is 400 μmol / L. At these concentrations, an optimal solution is achieved that balances induction effect and cell stability, efficiently reproducing liver lipid metabolism-related characteristics.

[0031] In some specific embodiments of the present invention, palmitic acid and oleic acid are added to organoid amplification culture medium in a certain proportion and cultured for 5 days to obtain a fatty liver organoid model.

[0032] Based on the technical solution of the present invention, the present invention has the following beneficial effects compared with the prior art:

[0033] This invention provides a method for establishing a liver organoid model. The method is simple, low-cost, and produces organoids with clear boundaries and uniform morphology, with a high yield. Compared with existing construction methods, the organoid yield is significantly increased (by 40%).

[0034] The liver organoid model constructed using the method provided in this invention can also be used to construct a fatty liver model. The constructed fatty liver model is highly responsive to drugs for treating fatty liver, enabling high-throughput screening of drugs for treating fatty liver and providing a basis for the clinical screening of novel anti-inflammatory drugs. Attached Figure Description

[0035] Figure 1 These are images showing the results of cell morphology observation under different digestion methods; among them, Figure 1 In the figure, A represents the morphological observation results of cells after treatment with digestion method 1; Figure 1 In the figure, B represents the morphological observation results of cells after treatment with digestion method 2.

[0036] Figure 2 This is a morphological image of a human bile duct organoid model after 10 days of culture during the amplification stage in an embodiment of the present invention.

[0037] Figure 3 This is a morphological diagram of a human liver organoid model after 15 days of culture during the differentiation stage in an embodiment of the present invention.

[0038] Figure 4 This is a graph showing the statistical results of the number of organoids obtained under different digestion methods.

[0039] Figure 5 This is a graph showing the statistical results of ATP production in organoids obtained under different digestion methods.

[0040] Figure 6 BODIPY staining image of lipid droplets after treatment with a 50μm small molecule compound from traditional Chinese medicine in a human fatty liver organoid model.

[0041] Figure 7 The image shows the cytokine levels in a human fatty liver organoid model after treatment with a 50μm small molecule compound from traditional Chinese medicine; among them, Figure 7 In the figure, A represents the statistical results of total cholesterol levels in different treatment groups; Figure 7 In the figure, B represents the statistical results of triglyceride levels in different treatment groups; Figure 7 In the figure, C represents the statistical results of IL-6 factor levels in different treatment groups; Figure 7 In the figure, D represents the statistical results of TNF-α factor levels in different treatment groups.

[0042] Figure 8 The image shows the cytokine levels in a human fatty liver organoid model after treatment with different concentrations of baicalin (a); among them, Figure 8 In the figure, A represents the statistical results of total cholesterol levels after treatment with different concentrations of baicalin; Figure 8 In the graph, B represents the statistical results of triglyceride levels. Figure 8 In the figure, C represents the statistical results of IL-6 factor levels after treatment with different concentrations of baicalin; Figure 8 In the figure, D represents the statistical results of TNF-α factor levels after treatment with different concentrations of baicalin.

[0043] Figure 9 The image shows the cytokine levels in a human fatty liver organoid model after treatment with different concentrations of dihydroberberine (b); among them, Figure 9 In the figure, A represents the statistical results of total cholesterol levels after treatment with different concentrations of dihydroberberine; Figure 9 In the figure, B represents the statistical results of triglyceride levels after treatment with different concentrations of dihydroberberine; Figure 9 In the figure, C represents the statistical results of IL-6 factor levels after treatment with different concentrations of dihydroberberine; Figure 9 In the figure, D represents the statistical results of TNF-α factor levels after treatment with different concentrations of dihydroberberine.

[0044] Figure 10 The image shows the cytokine levels in a human fatty liver organoid model after treatment with different concentrations of hyperoside (c); among them, Figure 10 In the figure, A represents the statistical results of the total cholesterol level of hyperoside after different concentrations of treatment. Figure 10 In the figure, B represents the statistical results of triglyceride levels after treatment with different concentrations; Figure 10 In the figure, C represents the statistical results of IL-6 factor levels after treatment with different concentrations; Figure 10 In the figure, D represents the statistical results of TNF-α factor levels after treatment with different concentrations. Detailed Implementation

[0045] The following description is based on specific embodiments.

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0047] The following description is based on specific embodiments.

[0048] The normal human liver tissue samples used in this embodiment were obtained from adjacent normal tissue removed during surgery at the People's Hospital of Longgang District, Shenzhen (verified by pathology). The acquisition of all samples was approved by the ethics review committee (approval number: 2024018), and the principle of informed consent was strictly followed.

[0049] Example 1: Method for establishing a human liver organoid model

[0050] This embodiment provides a method for establishing a human liver organoid model, as detailed below:

[0051] 1. Culture human bile duct 3D organoids and establish human bile duct organoid models.

[0052] (1) Sample acquisition and pretreatment: Normal liver tissue specimens were immediately placed in 4℃ pre-cooled epithelial organoid basal culture medium (manufacturer: Bozhen Biotechnology Co., Ltd., catalog number: B213151) and repeatedly rinsed until the supernatant was clear and free of impurities. The liver tissue specimens were then transferred to 7cm cell culture dishes, epithelial organoid basal culture medium was added, and the tissue was cut into 1-2mm pieces with surgical scissors. 3Fragments. Use a pipette to transfer the tissue block into a 50 mL centrifuge tube. After gravity sedimentation, aspirate the supernatant, add 4°C pre-cooled epithelial organoid basal culture medium, and resuspend 3-5 times until the supernatant is clear and free of impurities.

[0053] (2) Preparation of bile duct organoid suspension: The supernatant was removed, and the cells were digested using different digestion methods (digestion method 1 and digestion method 2). After digestion, the morphology of the organoids was observed in open field to confirm the appropriate digestion method. The digested tissue-containing solution was collected and filtered through a 100 μm filter to remove undigested tissue. The filtrate was collected in a clean 50 ml centrifuge tube, centrifuged at 4 °C and 250 g for 3 minutes, 1 mL of erythrocyte lysis buffer was added, and the cells were lysed at room temperature for 3 minutes, followed by another centrifugation. The organoid pellet was resuspended in epithelial organoid basal culture medium and washed twice by centrifugation at 4 °C and 250 g for 3 minutes to thoroughly remove digestion residues and obtain bile duct organoid suspension.

[0054] Digestion Method 1: After removing the supernatant, add 0.1% type IV collagenase sequentially, and digest at 37°C with shaking for 5 minutes; then add 0.2% type IV collagenase, and digest at 37°C with shaking for 5 minutes; finally, add 0.3% type IV collagenase, and digest at 37°C with shaking for 10 minutes. Observe under a microscope until a large number of cell clusters appear, then stop digestion. Add fetal bovine serum to a final concentration of 2% to stop digestion.

[0055] Digestion Method 2: After removing the supernatant, add 0.3% type IV collagenase directly and digest at 37°C with shaking for 20 minutes. Observe under a microscope until a large number of cell clusters appear, then stop digestion. Add fetal bovine serum to a final concentration of 2% to stop digestion.

[0056] (3) 3D culture induction: According to the amount of organoids, add an appropriate volume of epithelial organoid basal culture medium to resuspend the precipitate, take 20 μL and put it on a blood cell counter, count the number of organoids in the 20 μL sample, until the density of organoids is resuspended to 5-20 complete organoids / μL.

[0057] Take 200 μL of bile duct organoid suspension and add an equal volume of undiluted matrix gel mixed organoids, then mix thoroughly by pipetting. Take 50 μL of the organoid suspension (Matrix gel mixture) and drop it into the center of the bottom of each well in a 96-well plate. Incubate upside down in a 37°C, 5% CO2 incubator for 10-30 minutes until a solid droplet forms.

[0058] (5) Amplification culture: Add 200 μL of room temperature prepared organoid amplification medium to each well, and add sterile PBS to the other uninoculated wells to ensure humidity during culture. Cover the culture plate. When the organoid volume reaches 200-300 μm, aspirate the amplification medium and slowly add 600 μL of preheated differentiation medium along the well wall. Replace with fresh medium every 2-3 days and culture at 37℃ and 5% CO2 for 7-14 days (10 days in this example).

[0059] The amplification culture medium was based on DMEM / F12, supplemented with 2% (v / v) B27 additive (manufacturer: Shanghai Yuanpei Biotechnology Co., Ltd.; catalog number: S440J7), R-spondin 1 at a final concentration of 200 ng / mL, Noggin at a final concentration of 100 ng / mL, epidermal growth factor (EGF) at a final concentration of 50 ng / mL, fibroblast growth factor 10 (FGF10) at a final concentration of 50 ng / mL, and oncostatin M at a final concentration of 10 ng / mL.

[0060] (6) Differentiation culture: After a 10-day amplification culture stage, the organoid amplification medium was replaced with organoid differentiation medium, and the differentiation culture was carried out at 37℃ and 5% CO2 for 15 days to finally obtain a human liver organoid model.

[0061] The differentiation culture medium was based on DMEM / F12, supplemented with 2% (v / v) B27 additive, 1% (v / v) insulin-transferrin-selenium complex (ITS), hepatocyte growth factor (HGF) at a final concentration of 30 ng / mL, oncostatin M at a final concentration of 20 ng / mL, fibroblast growth factor 4 (FGF4) at a final concentration of 5 ng / mL, bone morphogenetic protein 7 (BMP7) at a final concentration of 10 μM, A83-01 at a final concentration of 5 μM, DAPT at a final concentration of 0.5 μM, and dexamethasone at a final concentration of 0.5 μM.

[0062] 2. Experimental Results

[0063] (1) Observation results of cell morphology after different digestion methods

[0064] like Figure 1As shown, the tissue initially exhibited mild dissociation under the initial action of low-concentration enzyme (0.1%), with intercellular connections gradually loosening; at medium concentration (0.2%), it further dissociated into smaller cell clusters; and at high concentration (0.3%), it ultimately formed organoids with clear boundaries and uniform morphology. Under bright-field microscopy, the cell clusters showed high integrity, low fragmentation rate, and smooth organoid surfaces without obvious signs of mechanical damage. Prolonged action by high-concentration enzyme (0.3%) led to rapid tissue disintegration, with cell clusters of varying sizes and a significant increase in fragmentation. Some organoids showed blurred edges, and even cell membrane rupture or vacuolation, indicating structural damage caused by over-digestion.

[0065] (2) Identification of human bile duct organoids and human liver organoids

[0066] The morphology of the established human bile duct organoid model was observed and photographed after 10 days of culture during the expansion phase. The results are as follows: Figure 2 As shown, the expanded human bile duct organoids are larger in size and exhibit a hollow sac-like structure.

[0067] The morphology of the established human liver organoid model was observed and photographed after 15 days of culture during the differentiation stage. The results are as follows: Figure 3 As shown, it can be seen that after differentiation, human liver organoids gradually transform into solid grape-like clusters, with the internal lumen disappearing and cell density significantly increasing.

[0068] (3) Identification of organoid quantity

[0069] like Figure 4 As shown, the gradient method, by avoiding acute stress through stepwise enzymatic digestion, maintains the intercellular signaling network, thereby improving organoid formation efficiency and structural stability. Organoid yield increased significantly (by 40%). An ATP assay kit was used to observe ATP production in organoids.

[0070] (4) ATP production detection after different treatments

[0071] ATP was measured the day after digestion and plating. Figure 5 As shown, the gradient method, by avoiding acute stress through stepwise enzymatic digestion, maintains the intercellular signaling network, thereby improving organoid formation efficiency and structural stability. Organoid yield increased significantly (by 40%). An ATP assay kit was used to observe ATP production in organoids.

[0072] Example 2: Application of human liver organoid models in the screening of drugs for the treatment of fatty liver

[0073] 1. Construction of human fatty liver organoid model:

[0074] The human liver organoids constructed in Example 1 were induced and treated with free fatty acids (FFA) to construct a fatty liver organoid model. The specific construction method is as follows:

[0075] (1) The human liver organoids constructed in Example 1 were digested from the matrix gel and gently blown to form individual organoids;

[0076] (2) Resuspend the organoids in pre-cooled culture medium, and take 200 μL of organoid suspension (5-20 complete organoids / μL), mix it with matrix gel (such as Matrigel) at a volume ratio of 1:1, and drop it into the well plate to form gel droplets;

[0077] (3) Slowly add organoid culture medium along the well wall to avoid dispersing the matrix droplets. Place the culture plate in a 37℃, 5% CO2 incubator and incubate until the organoids grow to a suitable size of 200-300μm and density (merging degree reaches 50%-60%) before induction.

[0078] (4) Thaw the prepared FFA stock solution (composed of palmitic acid and oleic acid), vortex mix well, and add it to the organoid complete culture medium according to the calculated volume. Discard the old culture medium and add induction medium containing FFA (based on DMEM / F12 medium, with 2% (v / v) B27 additive, 1% (v / v) insulin-transferrin-selenium complex (ITS), hepatocyte growth factor (HGF) at a final concentration of 30 ng / mL, oncostatin M at a final concentration of 20 ng / mL, fibroblast growth factor 4 (FGF4) at a final concentration of 5 ng / mL, bone morphogenetic protein 7 (BMP7) at a final concentration of 10 μM, A83-01 at a final concentration of 5 μM, and DAPT at a final concentration of 0.5 μM). μM dexamethasone, palmitic acid and oleic acid at final concentrations of 200 μmol / L and 400 μmol / L respectively);

[0079] (5) The organoids were returned to the incubator and induced for 7 consecutive days. During this period, the fresh induction medium containing FFA was replaced every 1-2 days to obtain a human fatty liver organoid model. Then, the human fatty liver organoid model was validated using drugs for treating fatty liver.

[0080] 2. Screening of different drugs for treating fatty liver at the same concentration

[0081] (1) Experimental grouping and treatment

[0082] The human fatty liver organoid model constructed in step 1 was randomly divided into 4 groups: model control group (FFA group), baicalin treatment group (group a), dihydroberberine treatment group (group b), and hyperoside treatment group (group c), with 3 replicates in each group.

[0083] Organoid culture medium containing 50 μmol / L baicalin, 50 μmol / L dihydroberberine, and 50 μmol / L hyperoside (drugs dissolved in DMSO, final DMSO concentration ≤0.1%) was added to groups a, b, and c, respectively. The model control group was given organoid culture medium containing an equal amount of DMSO. The cultures were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0084] (2) Detection indicators and methods

[0085] 1) Observation of lipid droplet formation: After culture, the culture medium was discarded, the organoids were washed three times with PBS, and BODIPY493 / 503 staining solution (final concentration 1 μg / mL) was added. The organs were incubated at 37°C in the dark for 30 minutes. After washing twice with PBS, the lipid droplet fluorescence signal was observed and photographed under a fluorescence microscope to analyze the number and area of ​​lipid droplet formation.

[0086] 2) Detection of inflammatory factor levels: Culture supernatant from each group was collected, and the levels of inflammatory factors (such as TNF-α, IL-6, and IL-1β) were detected using ELISA kits (TNF-α, manufacturer: abcam, catalog number: ab181421; IL-6, manufacturer: abcam, catalog number: ab278117; IL-1β, manufacturer: abcam, catalog number: ab260072). The operation was strictly performed in accordance with the kit instructions.

[0087] 3. Screening of different concentrations of the same drug for treating fatty liver

[0088] (1) Experimental grouping and treatment

[0089] Using the human fatty liver organoid model constructed in step 1, experiments were set up for three drugs: baicalin, dihydroberberine, and hyperoside. Five groups were set up for each drug: model control group (FFA group), 25 μmol / L treatment group, 50 μmol / L treatment group, 100 μmol / L treatment group, and 200 μmol / L treatment group. Each group had three replicates.

[0090] Organoid culture medium containing the corresponding concentration of drug (final DMSO concentration ≤ 0.1%) was added to each concentration treatment group, while organoid culture medium containing an equal amount of DMSO was added to the model control group. The samples were cultured at 37°C and 5% CO2 for 48 hours.

[0091] (2) Detection indicators and methods

[0092] Culture supernatants and organoids from each group were collected. The levels of inflammatory factors were detected using ELISA kits (TNF-α, manufacturer: abcam, catalog number: ab181421; IL-6, manufacturer: abcam, catalog number: ab278117; IL-1β, manufacturer: abcam, catalog number: ab260072). At the same time, the total cholesterol and triglyceride contents in the organoids were detected using biochemical assay kits (triglycerides: manufacturer: Shanghai ELISA, catalog number: ml076637; total cholesterol: manufacturer: Shanghai ELISA, catalog number: ml092734). All operations were performed in accordance with the corresponding instructions.

[0093] 4. Experimental Results

[0094] (1) Screening and detection results of different drugs for treating fatty liver at the same concentration

[0095] The results are as follows Figure 6 and Figure 7 As shown. Figure 6 The formation of lipid droplets after treatment with 50 μmol / L drugs (fluorescence microscopy images and quantitative analysis). Figure 7 The results show the levels of inflammatory factors in each group.

[0096] As shown in the figure, compared with the model control group (FFA group), the number and area of ​​lipid droplets in groups a, b, and c were significantly reduced, and the levels of total cholesterol and free fatty acids were significantly decreased (P<0.05). Simultaneously, the levels of inflammatory factors (TNF-α, IL-6, IL-1β) in each group were significantly lower than those in the model control group (P<0.05). These results indicate that the human fatty liver organoid model constructed in this invention has good sensitivity to different drugs for treating fatty liver at the same concentration and can be used for screening drugs for fatty liver treatment.

[0097] (2) Screening and detection results of the same drug for treating fatty liver at different concentrations

[0098] The results are as follows Figure 8 , Figure 9 , Figure 10 The figures show the results of inflammatory factor levels and lipid content detection after treatment with different concentrations of baicalin (a), dihydroberberine (b), and hyperoside (c).

[0099] As shown in the figure, compared with the model control group (FFA group), all concentrations of the three drugs significantly reduced the levels of total cholesterol, triglycerides, and inflammatory factors (TNF-α, IL-6, IL-1β) in the organoids (P<0.05); and as the drug concentration increased from 25 μmol / L to 200 μmol / L, the reduction in these indicators gradually increased, showing a concentration-dependent relationship (P<0.05). These results indicate that the fatty liver organoid model constructed in this invention has good sensitivity to different concentrations of drugs for treating fatty liver and can be used for dose-dependent screening of small molecule traditional Chinese medicine drugs for fatty liver treatment.

[0100] In summary, this invention provides a method for establishing a liver organoid model. This method is simple, low-cost, and produces organoids with clear boundaries, uniform morphology, and high yield. The liver organoid model constructed using this method can also be used to construct a fatty liver model. The resulting fatty liver model responds sensitively to drugs for treating fatty liver, enabling high-throughput screening of these drugs and providing a basis for the clinical screening of novel anti-inflammatory drugs.

[0101] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0102] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for establishing a liver organoid model, characterized in that, include: The obtained liver tissue samples were pretreated to obtain a mixture containing liver tissue-derived cell clusters; The mixture containing liver tissue-derived cell clusters was subjected to gradient digestion. After digestion, the undigested tissue was filtered out, the filtrate was collected, lysed, and centrifuged to obtain bile duct organoids. The bile duct organoids were induced and cultured to obtain liver organoid models; The gradient digestion method involves sequentially digesting the mixture containing liver tissue-derived cell clusters with type IV collagenase at mass concentrations of 0.1%, 0.2%, and 0.3% for 5 to 10 minutes.

2. The method for establishing as described in claim 1, characterized in that, The induction culture method includes: mixing the bile duct organoids with matrix gel in a certain proportion, forming solid droplets after the matrix gel solidifies, followed by amplification culture and differentiation culture, and finally forming a liver organoid model.

3. The method for establishing as described in claim 2, characterized in that, The amplification culture lasts for 7 to 14 days; the differentiation culture lasts for 10 to 15 days.

4. The method for establishing as described in claim 2, characterized in that, The bile duct organoids were mixed with matrix gel at a volume ratio of 1:1-3.

5. A liver organoid model, characterized in that, It is constructed by the method described in any one of claims 1 to 5.

6. The application of the liver organoid model as described in claim 5 in the construction of liver disease models.

7. The application as described in claim 6, characterized in that, The liver disease model mentioned is a fatty liver disease model.

8. The application of the liver organoid model as described in claim 5 in drug screening for fatty liver.

9. A method for constructing a fatty liver organoid model, characterized in that, include: The fatty liver organoid model was obtained by inducing treatment with free fatty acids as described in claim 5.

10. The construction method as described in claim 9, characterized in that, The induction treatment included treating the liver organoid model with an induction medium containing palmitic acid and oleic acid; The free fatty acids include palmitic acid and oleic acid; in the induction medium, the final concentration of palmitic acid is 200 μmol / L to 1000 μmol / L, and the final concentration of oleic acid is 200 μmol / L to 1000 μmol / L.

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