Method for constructing mature liver organoid with high expression of NTCP and HBV infection model

Abstract

The invention provides a method for constructing a mature liver organoid with high expression of NTCP and an HBV infection model, the maturity of the liver organoid is effectively improved by adding 0.01-0.5 [mu] M of traditional Chinese medicine micromolecule saikoside A (SSA) for collaborative differentiation, and the mature liver organoid with high expression of NTCP receptors is obtained. The HBV infection model established on the basis improves the invasion efficiency and replication level of the virus, and provides a reliable in-vitro platform for researching the interaction between HBV and host cells and evaluating the curative effect of traditional Chinese medicine micromolecules or antiviral drugs.

Description

Technical Field

[0001] This invention belongs to the field of biological research, and in particular relates to a method for constructing a mature liver organoid with high expression of NTCP and an HBV infection model. Background Technology

[0002] Hepatitis B virus (HBV) infection is one of the most important chronic viral liver diseases worldwide. Extensive epidemiological data show that persistent HBV infection is closely associated with chronic hepatitis, progression of liver fibrosis, cirrhosis, and hepatocellular carcinoma, placing a long-term and heavy burden on public health systems. Although vaccination and the use of some antiviral drugs have reduced new infections and viral replication levels to some extent, there is currently a lack of a radical strategy to completely eliminate covalently closed circular DNA (cccDNA), and a large number of patients remain chronically infected. Therefore, it is necessary to deeply understand the mechanisms of HBV invasion, replication, and host interaction, and to develop novel interventions based on this understanding, relying on stable, reproducible infection models that highly mimic the physiological state of the human liver.

[0003] However, HBV infection exhibits significant host specificity, efficiently infecting only humans and a very small number of higher primates, such as chimpanzees. Conventional mice and most laboratory animals are naturally insensitive to HBV, severely limiting the construction and application of in vivo models. While humanized liver mouse and tree shrew models have overcome species barriers to some extent, they still face challenges such as long modeling cycles, high costs, significant individual variability, and strict ethical restrictions, making it difficult to meet the needs of large-scale drug screening and mechanism research. Regarding in vitro models, primary human hepatocytes are limited in source and prone to dedifferentiation during in vitro culture. While commonly used liver cancer cell lines are readily available and easily expanded, they differ significantly from normal liver tissue in metabolic function, receptor expression, and immune-related pathways, only partially reflecting the HBV infection process. These limitations make the construction of humanized HBV infection models that more closely resemble the physiological state of the human liver a critical issue that urgently needs to be addressed.

[0004] The rise of organoid technology has provided a significant opportunity to establish a new generation of humanized in vitro infection models. Liver organoids, formed through three-dimensional culture of human pluripotent stem cells (hPSCs) or tissue-specific stem cells, can self-organize into liver-like structural units in vitro, expressing various liver marker genes and functionally related enzymes. They partially reproduce characteristics such as bile acid metabolism, drug metabolism, and polarity differentiation, providing a novel experimental platform for studying liver development, the pathogenesis of liver diseases, and personalized medicine. In the field of HBV research, liver organoids are expected to overcome the multiple limitations of traditional two-dimensional cell and animal models in terms of infection susceptibility, patient-specific responses, and long-term culture stability, becoming an important bridge connecting basic research and translational applications.

[0005] However, existing liver organoids still have significant limitations in constructing HBV infection models. Most differentiation protocols yield liver-like cells with limited maturity, whose overall function is closer to that of the fetal liver. These cells exhibit low expression levels of drug-metabolizing enzymes, bile acid transporters, and key invasion receptors (such as NTCP), resulting in low natural susceptibility to HBV, low infection efficiency, and low viral replication levels. There is an urgent need to develop a novel liver organoid model that possesses both higher maturity and stronger HBV susceptibility, providing a reliable platform for subsequent mechanistic studies and antiviral drug screening. Summary of the Invention

[0006] In view of this, the present invention aims to propose a method for constructing mature liver organoids with high NTCP expression and an HBV infection model. By promoting the development and maturation of hepatocytes in liver organoids through the small molecule of traditional Chinese medicine, saikosaponin A, the maturity of organoids is improved, while significantly increasing HBV infection susceptibility and replication level. Based on this, a new in vitro HBV infection model is constructed.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A method for constructing mature liver organoids with high NTCP expression: adding 0.01μM-0.5μM saikosaponin A (SSA) to the early hepatoid cell differentiation stage and the maturity maintenance stage of human pluripotent stem cells induced to differentiate into liver organoids, and culturing in a medium containing saikosaponin A for 5-10 days to finally obtain mature liver organoids.

[0008] SSA is a triterpenoid saponin compound derived from the traditional Chinese medicine Bupleurum chinense. SSA persists during the functional maturation of hepatocyte-like cells and the stable period of bile duct-like structures, thereby enhancing the overall metabolic capacity and structural integrity of organoids.

[0009] By adding SSA to the liver organoid culture system, the expression of genes related to bile acid synthesis and transport, and drug metabolism in liver organoids was increased, the formation of polar structures and tight junctions was promoted, and the expression levels of HBV-related receptors (such as NTCP) and cofactors were upregulated.

[0010] In addition, SSA has no significant effect on promoting maturation if the concentration is below the above level, and can damage cells if the concentration is above the above level.

[0011] Furthermore, during the maturation maintenance stage, a maturation medium was used for cultivation. In addition to saikosaponin A, the maturation medium also included glutamine at a volume concentration of 2.5%-5%, 5 ng / mL-20 ng / mL of serotonin M, and 0.2 μM-1.0 μM of dexamethasone in HepatoZYME-SFM medium.

[0012] Furthermore, the concentration of saikosaponin A was 0.05 μM-0.1 μM.

[0013] Furthermore, the method for constructing mature liver organoids with high NTCP expression specifically includes the following steps: (1) Endoderm differentiation stage: Based on RPMI 1640, add 20% mTeSR™ Plus, 5% insulin-free B27, 100-200 ng / ml cytokine Activin A, and 4-40 ng / ml cytokine BMP4, and culture for 1-5 days, changing the culture medium daily. (2) Differentiation stage of hepatic progenitor cells: Based on RPMI 1640, add 20% mTeSR™ Plus, 5% insulin-containing B27, 10-20 ng / ml cytokine FGF4 and 5-20 ng / ml cytokine BMP2, and culture for 5-8 days; (3) Early stage of hepatoid cell differentiation: Then replace it with RPMI 1640 as the base, with a volume concentration of 20% mTeSR™ Plus, a volume concentration of 5% B27 containing insulin, 10-40 ng / ml of cytokine HGF, 10-40 ng / ml of cytokine FGF7 and 0.01 μM-0.5 μM saikosaponin A (SSA), and continue to culture for 4-6 days; (4) Maturity maintenance stage: Culture in mature culture medium for 5-10 days. Mature culture medium consists of HepatoZYME-SFM medium with a volume concentration of 2.5%-5% glutamine, 5 ng / mL-20 ng / mL serotonin M, 0.2 μM-1.0 μM dexamethasone, and 0.01 μM-0.5 μM saikosaponin A (SSA).

[0014] This invention also provides a method for constructing an HBV infection model, the method comprising the following steps: I. Using the method described above, obtain mature liver organoids; II. The mature liver organoids obtained in step I were pretreated with a maturation culture medium containing saikosaponin A to further stabilize the polar structure and maintain high levels of NTCP receptor expression. III. Incubate HBV virus particles with pretreated mature liver organoids to establish infection; IV. The infected mature liver organoids were cultured in a maintenance medium that supports HBV replication to construct an HBV infection model.

[0015] Furthermore, the pretreatment time in step II is 12-48 hours, and the concentration of saikosaponin A is 0.01μM-0.5μM; the culture medium used for pretreatment is mature culture medium.

[0016] Furthermore, in step III, the method for preparing HBV virus particles is as follows: culture a cell line that stably secretes HBV, collect the cell culture supernatant, filter it, and then enrich it by sucrose density gradient ultracentrifugation.

[0017] Furthermore, the infectious dose of HBV virus particles is 1000-3000 GE / cell, and the co-incubation conditions are: incubation at 37°C for 18-36 hours.

[0018] Furthermore, the specific preparation method of HBV virus particles is as follows: Select a stable HBV-secreting cell line (such as HepAD38) for expanded culture, collect the supernatant at an appropriate time, filter to remove cell debris, enrich the virus by sucrose density gradient ultracentrifugation, discard the supernatant, resuspend the precipitate in a suitable buffer or low-protein medium (such as Opti-MEM), aliquot and store at low temperature.

[0019] Infection dose setting: HBV DNA was extracted from the viral suspension, and the HBV DNA copy number was determined by real-time PCR. Based on this, the viral particle titer was calculated for subsequent infection dose setting.

[0020] Furthermore, in step IV, the maintenance medium is a mature medium that does not contain dexamethasone, and the maintenance medium contains saikosaponin A at a concentration of 0.01 μM-0.5 μM. The culture is carried out in the maintenance medium for 7-20 days.

[0021] In step IV, small molecule SSAs of traditional Chinese medicine are retained as needed to maintain organoid maturity and stability, while avoiding the selection of components with obvious direct antiviral activity that may interfere with model establishment.

[0022] The present invention also provides an HBV infection model constructed by the method described above.

[0023] This invention also provides an application of the HBV infection model described above in screening anti-HBV drugs.

[0024] Compared with existing technologies, the method for constructing mature liver organoids and HBV infection models with high NTCP expression described in this invention has the following advantages: This invention utilizes a synergistic differentiation technique with the small molecule saikosaponin A (SSA) from traditional Chinese medicine to effectively enhance the maturity of liver organoids, resulting in higher levels of metabolic enzyme activity, bile acid transport, and HBV receptor-related gene expression. The HBV infection model established based on this technology improves viral invasion efficiency and replication levels, providing a reliable in vitro platform for studying the interaction between HBV and host cells, and evaluating the efficacy of small molecule traditional Chinese medicine or antiviral drugs. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 A schematic diagram illustrating the construction of a mature liver organoid with high NTCP expression and an HBV infection model; Figure 2 Bright-field images of liver organoids, scale bar: 200 μm; (a) early liver organoids, (b) mature liver organoids; Figure 3 Bar chart showing the expression levels of hepatocyte functional gene ALB, cholangiocarcinoma gene CK19, liver metabolic enzyme-related genes CYP3A4 and CYP7A1, bile duct transport-related gene CFTR, and HBV infection receptor NTCP in the SSA-treated group and the control group (without SSA). *p<0.05, **p<0.01, ***p<0.001. Figure 4 Immunohistochemical staining images of biomarkers in liver organoids, scale bar: 20 μm; (a) is the hepatocyte functional gene ALB, (b) is the bile duct cell gene CK19; Figure 5 This is an immunofluorescence image of mature liver organoid hepatocyte markers HNF4α and NTCP co-stained in Example 1; Figure 6 This is an immunofluorescence image of mature liver organoid hepatocyte markers HNF4α and NTCP co-stained in Example 2; Figure 7 The results of PCR detection of HBV pgRNA levels on day 7 after infection with HBV in mature liver organoids are shown. *p<0.05, **p<0.01, ***p<0.001; Figure 8 The results of ELISA detection of HBeAg and HBsAg in the supernatant of mature liver organoids on day 7 after infection with HBV are shown. *p<0.05, **p<0.01, ***p<0.001; Figure 9 Bright field image of a mature liver organoid on day 7 after HBV infection; Figure 10 Immunofluorescence staining image showing significant expression of core and S proteins in mature liver organoids on day 7 after HBV infection. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 scope of protection of the present invention.

[0028] Example 1: Construction of a mature liver organoid with high expression of NTCP Starting with iPSCs (induced pluripotent stem cells), the cells are initially differentiated into hepatic progenitor cells using conventional methods. Subsequently, 0.01 μM-0.5 μM SSA is added to the culture medium for the early hepatoid cell differentiation stage and the maturation maintenance stage. The differentiation process is as follows: Figure 1 As shown, the following differentiation steps were all carried out in a humidified incubator at 37°C and 5% CO2. The reagents used in this method are shown in Table 1.

[0029] The specific steps are as follows: Stage 1: iPSCs differentiate into early liver organoids (1) Differentiate hPSCs 24-48h after passage.

[0030] (2) Endoderm differentiation: Based on RPMI 1640, 20% mTeSR™ Plus, 5% insulin-free B27, 150 ng / ml Activin A, and 5 ng / ml BMP4 were added. The culture was incubated for 5 days, with fresh medium added daily.

[0031] (3) Differentiation of hepatic progenitor cells: Based on RPMI 1640, 20% mTeSR™ Plus, 5% insulin-containing B27, 20 ng / ml cytokine FGF4, and 15 ng / ml cytokine BMP2 were added and cultured for 6 days.

[0032] (4) Early differentiation of hepatoid cells: Then, the culture medium was switched to RPMI 1640 as a base, and 0.05 μM of saikonin A (SSA), 20% mTeSR™ Plus, 5% insulin-containing B27, 40 ng / ml of cytokine HGF, and 40 ng / ml of cytokine FGF7 were added. The mixture was cultured for 5 days, thus obtaining early liver organoids. Figure 2 As shown in (a).

[0033] Phase 2: SSA treatment promotes liver organoid maturation (maturation maintenance phase) The early liver organoids obtained in stage 1 were cultured in a maturation medium for another 8 days to obtain mature liver organoids, such as... Figure 2 As shown in (b). Change the culture medium every 2 days.

[0034] The maturation medium consisted of HepatoZYME-SFM medium supplemented with 0.05 μM SSA, 5% glutamine (v / v), 5 ng / ml serotonin M, and 0.2 μM dexamethasone. Continuous culture resulted in more compact and complex organoid structures.

[0035] Figure 2 The image shows a bright-field image of a liver organoid. It can be seen from the image that the liver organoid structure is more compact after SSA treatment, the morphology of hepatocytes is clearer, and they exhibit mature multinucleated, polygonal structures with regular polarity arrangement.

[0036] To demonstrate the importance of SSA, a control group was set up for comparison. The control group did not add SSA during the above culture process, and all other aspects were the same.

[0037] After culture, liver organoids were collected for the following analyses: qRT-PCR: The expression levels of hepatocyte functional genes ALB, cholangiocarcinocyte gene CK19, liver metabolic enzyme-related genes CYP3A4 and CYP7A1, bile duct transport-related gene CFTR, and HBV infection receptor NTCP in mature liver organoids obtained from Example 1 (i.e., the SSA-treated group) and the control group were detected. Results are as follows: Figure 3 As shown, the expression levels of the aforementioned genes in the SSA-treated group were significantly higher than those in the control group, indicating that SSA increased the expression of genes related to bile acid synthesis and transport, and drug metabolism in liver organoids, especially upregulating the expression level of HBV-related receptors (such as NTCP). This demonstrates the importance of SSA in promoting hepatocyte functional maturation and high NTCP expression. NTCP is a core parameter for constructing HBV models and directly affects the accuracy of the model. The NTCP expression level determines the infection efficiency; higher NTCP expression levels lead to faster viral entry and more active replication, resulting in a better HBV infection model.

[0038] Immunohistochemistry: Expression of ALB (hepatocytes) and CK19 (cholangiocarcinocytes) markers in liver organoids, as shown in... Figure 4 As shown, this concentration of SSA intervention does not affect the lineage development of liver organoids and bile duct cells.

[0039] Immunofluorescence: Expression of NTCP receptor in liver organoids after SSA treatment, as shown below. Figure 5 As shown, SSA can promote the high expression of NTCP protein in hepatocytes (HNF4α-labeled) after liver organoid maturation.

[0040] Conclusion: This embodiment demonstrates that adding SSA during the construction of liver organoids can effectively promote the functional maturation of hepatocytes and highly express the HBV infection-related receptor NTCP, successfully constructing highly mature HBV-susceptible liver organoids.

[0041] Example 2 The specific operating procedure is the same as in Example 1, but the concentration of SSA is 0.5 μM.

[0042] like Figure 6 As shown, mature liver organoids with high expression of NTCP protein were eventually obtained.

[0043] Example 3: Construction of an HBV infection model Using mature liver organoids with high NTCP receptor expression prepared in Example 1, an in vitro infection model of HBV susceptibility and active replication was established, providing a reliable human in vitro platform for subsequent basic research and efficacy evaluation of HBV.

[0044] The specific construction steps are as follows: I. Using the method of Example 1, mature liver organoids with high expression of NTCP protein were obtained; II. Pre-infection treatment: Approximately 24 hours before planned HBV infection, the culture medium for mature liver organoids was replaced with the aforementioned fresh mature culture medium containing 0.05 μM SSA to further stabilize the polar structure of the organoids and HBV receptor expression.

[0045] During pretreatment, maintain standard culture conditions (37°C, 5% CO2) to allow the organoids to adapt to subsequent infection procedures.

[0046] III. HBV vaccination and infection establishment: 1. Virus preparation: 1.1 Cell lines and culture: (1) Select the HepAD38 cell line that stably secretes HBV and seed it in a conventional adherent culture flask or culture dish. The culture conditions are 37℃ and 5% CO2.

[0047] (2) Use a suitable basal culture medium containing tetracycline or its analogues to maintain the normal growth of cells. When the cell confluence reaches about 70%-90%, prepare for induction.

[0048] 1.2 Virus production induction: (1) Discard the original culture medium and replace it with a pre-warmed, tetracycline-free induction culture medium to relieve the inhibition of HBV replication and thus induce the production of a large amount of HBV.

[0049] (2) Continue to culture HepAD38 cells for 5-7 days. During this period, fresh induction medium can be added every 2 days as needed to maintain cell status and virus production.

[0050] 1.3 Supernatant Collection and Pretreatment: (1) On days 5-7 after induction, collect all the supernatant from the cell culture flask into centrifuge tubes and store them at 4°C.

[0051] (2) Remove cells and large debris by low-speed centrifugation (3000 rpm, 10 minutes) and collect the supernatant.

[0052] (3) The supernatant was filtered through a 0.45 μm filter membrane to further remove residual cells and particles, and a clear supernatant was obtained.

[0053] 1.4. Virus enrichment by sucrose density gradient ultracentrifugation: (1) Prepare a 20% and 50% sucrose discontinuous gradient in an ultracentrifuge tube, preferably using sterile PBS or a suitable buffer solution.

[0054] (2) Slowly add the filtered supernatant to the top of the sucrose gradient in layers to avoid mixing the gradient interface.

[0055] (3) After balancing the centrifuge tubes, place them in a pre-cooled ultracentrifuge rotor and centrifuge at 4°C and 25,000 rpm for about 16 hours to enrich HBV virus particles.

[0056] 1.5 Virus recovery and dialysis: (1) After centrifugation, carefully remove the centrifuge tube, observe the sucrose gradient interface under appropriate light, and transfer the turbid white virus containing the virus to a new centrifuge tube.

[0057] (2) Dialyze or dilute-centrifuge the virus strip with PBS or other suitable buffer to remove sucrose and obtain a relatively purified HBV virus suspension.

[0058] 1.6 Virus Preservation and Quantification: (1) According to the experimental needs, the virus suspension is divided into equal volumes into small-capacity cryovials to avoid frequent freezing and thawing. Each tube is for single use only.

[0059] (2) Store at -80℃ for a long time.

[0060] (3) Take one portion of the virus suspension for titer determination: extract HBV DNA from the virus suspension, determine the HBV DNA copy number using quantitative real-time PCR (qPCR), and calculate the genome equivalent (GE) concentration of the virus particles (in GE / mL) as the basis for subsequent infection dose setting.

[0061] 2. Virus inoculation and co-incubation: 2.1 Virus inoculation: (1) Calculate the total HBV virus dose required based on the number of cells in the organoids to be infected, and use the infection doses of 1000GE / cell, 2000GE / cell, and 3000GE / cell as reference infection doses.

[0062] (2) Calculate the required volume of virus suspension based on the known viral storage solution titer, and dilute the virus to the above-mentioned infection concentration using the prepared infection diluent (HepatoZYME-SFM+2%DMSO+4%PEG8000) to obtain virus infection solutions with different infection doses.

[0063] (3) Discard the original culture medium after pretreatment of liver organoids, add virus infection solution, and be careful not to generate a large number of bubbles so as not to affect the uniform contact of the organoids with the virus.

[0064] (4) Place the culture plate containing organoids and viruses in a 37°C, 5% CO2 incubator and incubate on a shaker at a low speed (about 50 rpm) for about 24 hours to increase the chance of contact between the virus and the surface and internal cells of the organoids.

[0065] (5) The morphology of organoids can be observed intermittently during incubation to ensure that there is no obvious disintegration or mass death.

[0066] 2.2 Washing to remove unbound viruses: (1) After incubation, remove the culture plate from the incubator and carefully discard the supernatant containing the virus.

[0067] (2) Slowly add pre-warmed PBS containing 1% BSA to each well to gently wash the organoids and avoid generating strong shear force.

[0068] (3) Repeat the washing steps a total of 3 times, carefully discarding the liquid to remove as many unbound virus particles as possible.

[0069] IV. Post-infection culture: (1) After washing, the organoids were recultured in a maintenance medium containing 0.05 μM SSA but without dexamethasone. The maintenance medium was a HepatoZYME-SFM medium system with 0.05 μM SSA, 5% glutamine, and 5 ng / ml tumor suppressin M.

[0070] (2) Replace the culture medium every 1-2 days after infection, while retaining a certain proportion of the old supernatant to maintain the environmental stability of the virus and secretory factors.

[0071] (3) On the 7th day after infection, some supernatant and organoid samples were collected to detect HBV antigen, nucleic acid and organoid function indicators to evaluate whether the infection was successful and its stability.

[0072] Through the above steps, based on the SSA-promoted maturation of multi-lineage liver organoids, an in vitro HBV infection model with high HBV infection efficiency, active viral replication, and long-term stable maintenance can be obtained.

[0073] V. HBV Infection Efficiency Detection: Samples were collected on day 7 post-infection for the following tests: Organoid infection supernatant was collected, and the expression levels of HBeAg (hepatitis B e antigen) and HBsAg (hepatitis B surface antigen) were detected by ELISA. Total RNA was extracted from organoids using the TRIzol method, and the expression level of HBV pgRNA was detected by RT-qPCR. Organoids were fixed in 4% paraformaldehyde, and immunofluorescence analysis was performed using HBV core protein and S protein. HBV infection efficiency was analyzed by fluorescence microscopy.

[0074] The pgRNA level of HBV in liver organoids was detected by RT-qPCR, such as Figure 7 As shown; ELISA can detect HBeAg and HBsAg secreted after infection, such as... Figure 8 As shown, within the tested viral concentration gradient range, the infection efficiency increased with increasing viral load. Furthermore, at a maximum equivalent of 3000 GE / cell 7 days post-infection, HBV-infected liver organoids still exhibited good survival (see...). Figure 9 Most hepatocytes still retain clear morphology and cellular activity.

[0075] Figure 10 Immunofluorescence staining revealed significant expression of HBV core and HBV S proteins in liver organoids infected with HBV, indicating active HBV replication.

[0076] In summary, this invention successfully established a mature liver organoid with high NTCP expression and an HBV infection model. This model utilizes SSA to enhance organoid maturity and NTCP receptor expression, significantly improving HBV susceptibility and successfully supporting HBV infection and normal survival in vitro. It provides a highly realistic in vitro platform for simulating in vivo HBV infection and subsequent research.

[0077] Table 1 Relevant Reagents

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing mature liver organoids with high NTCP expression, characterized in that, The method involves adding 0.01 μM-0.5 μM saikosaponin A to the early hepatocyte differentiation stage and the maturation maintenance stage of human pluripotent stem cells induced to differentiate into liver organoids, and culturing in a medium containing saikosaponin A for 5-10 days to finally obtain mature liver organoids.

2. The method for constructing mature liver organoids with high NTCP expression according to claim 1, characterized in that, During the maturation maintenance phase, a maturation medium was used for cultivation. In addition to saikosaponin A, the maturation medium also included glutamine at a volume concentration of 2.5%-5%, 5 ng / mL-20 ng / mL of serotonin M, and 0.2 μM-1.0 μM of dexamethasone in HepatoZYME-SFM medium.

3. The method for constructing mature liver organoids with high NTCP expression according to claim 1, characterized in that, The concentration of saikosaponin A was 0.05 μM-0.1 μM.

4. A method for constructing an HBV infection model, characterized in that, The method includes the following steps: I. Obtaining mature liver organoids using the method of any one of claims 1-3; II. The mature liver organoids obtained in step I were pretreated with a culture medium containing saikosaponin A to further stabilize the polar structure and maintain high levels of NTCP receptor expression. III. Incubate HBV virus particles with pretreated mature liver organoids to establish infection; IV. The infected mature liver organoids were cultured in a maintenance medium that supports HBV replication to construct an HBV infection model.

5. The method for constructing an HBV infection model according to claim 4, characterized in that, The pretreatment time in step II is 12-48 hours, and the concentration of saikosaponin A is 0.01μM-0.5μM; the culture medium used for pretreatment is mature culture medium.

6. The method for constructing an HBV infection model according to claim 4, characterized in that, In step III, the method for preparing HBV virus particles is as follows: culture a cell line that stably secretes HBV, collect the cell culture supernatant, filter it, and then enrich it by sucrose density gradient ultracentrifugation.

7. The method for constructing an HBV infection model according to claim 4, characterized in that, In step III, the infection dose of HBV virus particles is 1000-3000 GE / cell, and the co-incubation conditions are: incubation at 37°C for 18-36 hours.

8. The method for constructing an HBV infection model according to claim 4, characterized in that, In step IV, the maintenance medium is a mature medium that does not contain dexamethasone, and the maintenance medium contains saikosaponin A at a concentration of 0.01 μM-0.5 μM. The culture is carried out in the maintenance medium for 7-20 days.

9. An HBV infection model constructed by the method described in any one of claims 4-8.

10. The application of the HBV infection model as described in claim 9 in screening anti-HBV drugs.

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