Method for constructing HBV infection model based on multilineage liver and gall organs

Abstract

The invention provides a construction method of an HBV (Hepatitis B Virus) infection model based on multilineage hepatobiliary organs, which comprises the following steps: differentiating human pluripotent stem cells to form immune cell-containing hepatobiliary organs for further maturation culture, and enabling the hepatobiliary organs to be mature in function and highly express NTCP (Natriuretic Therapeutic Cells) by using a maturation culture medium containing bile metabolism substrate cholesterol and deoxybear bile acid, so as to obtain the HBV infection model based on the multilineage hepatobiliary organs. And then infecting the mature organs by adopting an HBV virus strain generated by HepAD38 cells to construct an in-vitro model which is susceptible to HBV and can maintain an infection state for a long time. According to the construction method, the liver infection and immune interaction microenvironment can be simulated more truly, and a stable and reliable experimental platform is provided for HBV infection mechanism research and anti-HBV drug screening.

Description

Technical Field

[0001] This invention relates to the field of in vitro model construction, and in particular to a method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids. Background Technology

[0002] Hepatitis B (HBV) is a serious infectious disease caused by the hepatitis B virus. HBV infection remains a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, posing a significant threat to human health and imposing a huge social and economic burden. Currently, a key challenge in HBV research is the species-specific limitation of HBV infection. HBV primarily infects humans and a few higher primates such as chimpanzees, making it difficult to effectively establish infection models in commonly used laboratory animals such as mice. This species limitation greatly hinders research into the mechanisms of HBV infection and the development of new drugs. Therefore, establishing humanized HBV infection models has become a crucial issue in current basic HBV research and antiviral drug development.

[0003] In recent years, the development of stem cell technology and organoid culture technology has provided new solutions for studying the physiological and pathological state of the liver. Organoid technology, through the induced differentiation of human pluripotent stem cells (hPSCs) to construct liver organoids, can mimic the specific structure and function of the human liver to a certain extent in vitro. However, current liver organoids still have limited susceptibility to HBV infection and cannot truly reflect the complex functions and microenvironment of the human liver. Summary of the Invention

[0004] In view of this, the present invention aims to propose a method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids, which can reflect the liver microenvironment of infection and immune interaction, thereby providing a more precise platform for studying the HBV infection mechanism at the cellular and molecular levels; it helps to overcome the bottleneck of humanized hepatitis B virus infection models, promote the development of HBV-related drug research and clinical translation, and provide new ideas and tools for developing novel anti-HBV drugs.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids, comprising the following steps: S1. Inducing human pluripotent stem cells to differentiate into hepatobiliary organoids containing immune cells; S2. The hepatobiliary organoids obtained in S1 were cultured in a maturation medium containing cholesterol and deoxyursolic acid, which are the substrates of bile metabolism, to obtain mature multi-lineage hepatobiliary organoids. S3. An HBV infection model was constructed by infecting mature multilineage hepatobiliary organoids with HBV virus strains produced from HepAD38 cells.

[0006] Furthermore, immune cells include macrophages and / or NK cells.

[0007] The presence of immune cells contributes to the long-term survival of organoids after HBV infection.

[0008] Furthermore, the volume concentration of cholesterol, a bile metabolism substrate, was 1%-10%, the concentration of deoxyursolic acid was 0.1μM-1.0μM, and the culture time for S2 was 8-20 days.

[0009] Furthermore, the infection dose of HBV virus strains is 1000-3000 GE / cell, and the infection duration is 24h-48h.

[0010] GE stands for genome equivalent, and GE / mL is the genome equivalent concentration. When infecting cells, it is converted to GE / cell.

[0011] Furthermore, S3 also adds polyethylene glycol 8000 at a volume concentration of 1%-4% during infection.

[0012] Furthermore, after S3 infection, unbound viral particles were removed and the cells were maintained in hepatobiliary organoid maintenance medium, which included the basic component HCM medium and the key additive OSM at 5 ng / mL-20 ng / mL.

[0013] Furthermore, the formation of hepatobiliary organoids by S1 includes the following stages: endoderm and mesoderm differentiation, hepatoblast induction, co-differentiation of hepatocytes and bile duct cells, and hepatocyte maturation. Among them, the culture medium for the endoderm and mesoderm differentiation stage is based on the liver differentiation medium, with the addition of 2.5%-25% of stem cell culture medium mTeSR™ Plus and BMP4 differentiation factor 4-40 ng / mL to obtain hepatobiliary organoids containing immune cells.

[0014] Furthermore, the specific method of S1 is as follows: Phase 1: Endoderm and mesoderm are cultured in medium 1. Medium 1 includes basic components and key additives. The basic components include RPMI1640 with a volume concentration of 2% B27 (insulin-free). The key additives include mTeSR™ Plus with a volume concentration of 2.5%-25%, Activin A at 100-200 ng / mL, and BMP4 at 4-40 ng / mL. Phase 2: Hepatocytes were cultured in medium 2, which included basic components and key additives. The basic components included RPMI1640 with a volume concentration of 2% B27, and the key additives included mTeSR™ Plus at a volume concentration of 2.5%-25%, FGF4 at 10-20 ng / mL, and BMP2 at 5-20 ng / mL. Phase 3: Hepatocytes and bile duct cells were cultured in medium 3, which included basic components and key additives. The basic components included RPMI1640 with a volume concentration of 2% B27, and the key additives included mTeSR™ Plus at a volume concentration of 2.5%-25%, HGF at 10-40 ng / mL, and FGF7 at 10-40 ng / mL. Phase 4: Culture in medium 4 until hepatocytes mature. Medium 4 includes basic components and key additives. The basic components include HCM medium, and the key additives include 5 ng / mL-20 ng / mL OSM and 0.1 μM-1.0 μM dexamethasone.

[0015] Furthermore, the culture was carried out in culture medium 1 for 1-4 days, in culture medium 2 for 3-5 days, in culture medium 3 for 5-7 days, and in culture medium 4 for 3-5 days.

[0016] Compared with existing technologies, the method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids described in this invention has the following advantages: (1) The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids described in this invention optimizes differentiation conditions and culture system. Bile metabolism substrates cholesterol and deoxyursolic acid promote hepatocyte functional maturation and high NTCP expression, thereby achieving high susceptibility to HBV infection and long-term in vitro infection, presenting a typical liver fibrosis phenotype caused by hepatitis B virus infection.

[0017] (2) The use of specific differentiation factors in the method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids described in this invention can effectively promote the multi-lineage differentiation of hepatocytes and increase their susceptibility to HBV infection. At the same time, hepatobiliary organoids can functionally simulate the complex immune microenvironment and physiological functions of the liver in vivo, providing an ideal platform for the study of HBV infection mechanisms and the development of anti-HBV drugs. Attached Figure Description

[0018] 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 1The images show hepatobiliary organoids containing immune cells obtained in Example 1; (A) is an immunofluorescence staining image of NK cells (CD56+, CD11B+) in the hepatobiliary organoids; (B) is an immunofluorescence staining image of macrophages (CD68+, CD11B+) in the hepatobiliary organoids. Figure 2 The relative expression level of the HBV infection receptor NTCP gene in the hepatobiliary organoids obtained in the first and second steps of Example 1 was detected by qPCR. Figure 3 These are representative immunofluorescence images of NTCP protein expression in hepatobiliary organoids obtained in the first and second steps of Example 1. Figure 4 This is a representative immunofluorescence image of NTCP protein expression in hepatobiliary organoids obtained after the second step of optimization in Example 2; Figure 5 This is a representative immunofluorescence image of NTCP protein expression in hepatobiliary organoids obtained after the second step of optimization in Example 3; Figure 6 The results of HBV cccDNA, pgRNA, and vDNA detection on day 7 after hepatobiliary organoid infection; Figure 7 Representative immunofluorescence images showing significant expression of HBcAg and HBsAg on day 7 after hepatobiliary organoid infection, scale bar 100 μm; Figure 8 Representative bright-field images of hepatobiliary organoids 20 days after HBV infection, scale bar 100μm; Figure 9 The results of dynamic HBsAg detection in hepatobiliary organs infected with HBV within 20 days. Figure 10 The results of reinfecting the HBV-susceptible cell line HepG-puro-NTCP with progeny viruses isolated from the supernatant of infected hepatobiliary organoids; Figure 11 Bright field images and immunofluorescence images of collagen fibers stained with HBV-infected hepatobiliary organoids 20 days later are shown. The control group is the result of infection using hepatobiliary organoids obtained in step one of Example 1. HBV infection is the result of infection using mature hepatobiliary organoids cultured to day 30 of Example 1. Figure 12 This is a comparison of cccDNA, HBV pgRNA, HBeAg, and vDNA in the supernatant of organoid cultures after 7 days of maintenance culture with or without tenofovir intervention for HBV-infected hepatobiliary organoids in Example 5. Detailed Implementation

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0020] 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.

[0021] Example 1: Construction of mature hepatobiliary organoids I. Inducing human pluripotent stem cells to differentiate into hepatobiliary organoids containing immune cells; Based on the differentiation plan established by the team in the early stage ( J Hepatol, 2019, 70: 1145-1158; Stem Cells Int, 2022, 2022: 3222427. In the traditional endoderm-directed differentiation process, in addition to the commonly used liver differentiation medium, 2.5% to 25% of stem cell culture medium mTeSR™ Plus and BMP4 differentiation factor 4-40 ng / mL are added to induce the production of mesodermal and endoderm-directed cells, ensuring that multi-lineage immune cells derived from the mesoderm are obtained during later maturation. Figure 1 As shown, the hepatobiliary organoids obtained in this step contain NK cells and macrophages.

[0022] The specific plan is as follows: Table 1 Specific Differentiation Steps

[0023] II. Based on the culture medium from stage 4 above (including HCM, 20 ng / mL OSM, and 0.1 μM dexamethasone), 1% (v / v) cholesterol and 0.1 μM deoxyursolic acid were added to obtain a maturation culture medium. The hepatobiliary organoids obtained in step one were then cultured in this maturation culture medium for days 20, 25, 30, and 35. The expression of the HBV infection receptor NTCP in the hepatobiliary organoids obtained in step one and those obtained in step two on days 20, 25, 30, and 35 was then detected using qPCR and immunofluorescence. The results are as follows: Figure 2 and Figure 3 As shown.

[0024] Comparison reveals that hepatobiliary organoids cultured in the second step using a maturation medium containing bile metabolism substrates cholesterol and deoxyursolic acid exhibit sustained high NTCP expression, with the highest NTCP expression level observed on day 30. In contrast, the first step, without the use of a medium containing these bile metabolism substrates, fails to express NTCP at a high level, with an expression level almost zero. This demonstrates the importance of cholesterol and deoxyursolic acid in promoting hepatocyte functional maturation and high NTCP expression. NTCP is a core parameter in the construction of hepatitis B virus models, directly affecting the accuracy of the model. The NTCP expression level determines the infection efficiency; higher NTCP expression leads to faster viral entry and more active replication. Therefore, this invention also demonstrates that the method of this invention provides prerequisites and susceptibility prediction for hepatobiliary organoids infected with HBV.

[0025] Example 2 The specific operating procedure is the same as in Example 1, except that the volume concentration of cholesterol, the bile acid metabolism substrate, is 2% and the concentration of deoxyursolic acid is 0.5 μM.

[0026] like Figure 4 As shown, mature hepatobiliary organoids with high expression of NTCP protein were eventually obtained.

[0027] Example 3 The specific operating procedure is the same as in Example 1, except that the volume concentration of cholesterol, the bile acid metabolism substrate, is 10% and the concentration of deoxyursolic acid is 1.0 μM.

[0028] like Figure 5 As shown, mature hepatobiliary organoids with high expression of NTCP protein were eventually obtained.

[0029] Example 4: Construction of an HBV infection model Organoids: Mature multi-lineage hepatobiliary organoids obtained by culturing to day 30 using the method described in Example 1.

[0030] Virus preparation: HepAD38 cells were cultured to a suitable density, and the medium was replaced with tetracycline-free medium to induce a large-scale production of virus. The culture supernatant was collected after 5-7 days and filtered through a 0.45 μm filter to remove cell debris. HBV virus particles were enriched by ultracentrifugation at 4°C (25,000 rpm, 16 hours) using a 20% / 50% discontinuous sucrose density gradient. Viral bands were collected, sucrose was removed by dialysis with PBS, and the virus was concentrated, aliquoted, and stored at -80°C.

[0031] Inoculation: Based on the number of cells in the organoids to be infected, use infection doses of 100GE / cell, 1000GE / cell, and 3000GE / cell as reference infection doses. Dilute the virus to the above infection concentrations using the prepared infection diluent (including HCM, 2% DMSO, and 4% PEG8000). Incubate at 37°C and 5% CO2 with low-speed shaking (approximately 50 rpm) for 24 hours to ensure sufficient contact between the virus and the organoids.

[0032] Washing: After infection, gently wash the organoids three times with PBS containing 1% BSA to remove unbound viral particles.

[0033] Post-infection culture: Replace with hepatobiliary organoid maintenance culture medium (including HCM and 20 ng / mL OSM) for maintenance culture.

[0034] Model evaluation: The following tests were performed on day 7 post-infection: cccDNA, HBV pgRNA, and supernatant vDNA levels were detected by RT-PCR. Figure 6 As shown, the higher the infection dose, the more active the viral replication.

[0035] Immunofluorescence staining results as follows Figure 7 As shown, the study revealed significant expression of HBcAg (hepatitis B core antigen) and HBsAg (hepatitis B surface antigen) in the hepatobiliary organoids after infection, indicating active HBV replication and successful construction of the HBV infection model.

[0036] The ability to detect persistent chronic infections in hepatobiliary organoids: Bright field images of liver and biliary organoids infected with low-to-medium dose (1000 GE / cell) HBV 20 days later are shown below. Figure 8 As shown in the figure, the liver and gallbladder organoids remained active after 20 days.

[0037] Figure 9 The results showed that HBsAg in hepatobiliary organs infected with low to medium doses (1000 GE / cell) continued to be secreted within 20 days, indicating that multi-lineage hepatobiliary organs can maintain long-term stable HBV infection.

[0038] from Figure 10 It can be seen that the progeny viruses isolated from the supernatant of HBV-infected hepatobiliary organoids are infectious in HepG-puro-NTCP cells, indicating that the HBV-infected hepatobiliary organoid model supports the complete life cycle of the virus.

[0039] The above data supports the complete life cycle of viral genome cccDNA establishment, transcription, replication, and antigen secretion.

[0040] Furthermore, compared with the control group of hepatobiliary organoids not infected with HBV, HBV-infected hepatobiliary organoids showed a significant fibrotic pathological phenotype after 20 days post-infection, such as... Figure 11 As shown.

[0041] Thus, this invention has obtained the typical pathological phenotype of liver fibrosis caused by hepatitis B infection, indicating that the method of this invention can successfully construct an HBV infection model, and the survival period of organoids after HBV infection can be as long as 20 days.

[0042] Example 5: HBV infection model used for positive drug testing The HBV infection model constructed in Example 4 was used to perform a positive test for tenofovir, a commonly used antiviral drug in clinical practice. Tenofovir is a nucleoside reverse transcriptase inhibitor that can prevent the hepatitis B virus pregenomic RNA from being reverse transcribed into DNA.

[0043] Following the infection protocol in Example 4, mature hepatobiliary organoids were infected with an infection dose of 1000 GE / cell for 24 hours. The test group was cultured in hepatobiliary organoid maintenance medium supplemented with 5 μM tenofovir, while the control group was cultured in hepatobiliary organoid maintenance medium without tenofovir. The hepatobiliary organoid maintenance medium included HCM and 20 ng / mL OSM.

[0044] After 7 days of maintenance culture, cultures and supernatants of the two groups of organoids were collected, and changes in cccDNA and HBV pgRNA in the organoids and HBeAg and vDNA in the supernatant were detected (see [link to article]). Figure 12 The results showed that the production of HBeAg and vDNA in the culture supernatant was significantly inhibited by tenofovir, while there were no significant changes in ccc DNA and HBV pgRNA in organoids. This result is consistent with the mechanism by which tenofovir prevents the reverse transcription of hepatitis B virus pregenomic RNA into DNA.

[0045] The results of this embodiment demonstrate that the HBV-infected hepatobiliary organoids constructed in this invention can not only evaluate the efficacy of antiviral drugs, but also provide information on the drug action mechanism targeting specific stages of the disease life cycle.

[0046] 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 a model of HBV infection based on a multi-ethnic liver-biliary organoid, characterized by, The method includes the following steps: S1. Inducing human pluripotent stem cells to differentiate into hepatobiliary organoids containing immune cells; S2. The hepatobiliary organoids obtained in S1 were cultured in a maturation medium containing cholesterol and deoxyursolic acid, which are the substrates of bile metabolism, to obtain mature multi-lineage hepatobiliary organoids. S3. An HBV infection model was constructed by infecting mature multilineage hepatobiliary organoids with HBV virus strains produced from HepAD38 cells.

2. The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids according to claim 1, characterized in that, Immune cells include macrophages and / or NK cells.

3. The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids according to claim 1, characterized in that, The volume concentration of cholesterol, a bile metabolism substrate, was 1%-10%, and the concentration of deoxyursolic acid was 0.1μM-1.0μM. The culture time for S2 was 8-20 days.

4. The method of claim 1, wherein the method is performed by the method of claim 2. The infection dose of HBV virus strains is 1000-3000 GE / cell, and the infection duration is 24h-48h.

5. The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids according to claim 1, characterized in that, S3 also adds polyethylene glycol 8000 at a volume concentration of 1%-4% during infection.

6. The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids according to claim 1, characterized in that, After S3 infection, unbound viral particles were removed and the cells were maintained in hepatobiliary organoid maintenance medium, which consisted of the basic component HCM medium and the key additive OSM at 5 ng / mL-20 ng / mL.

7. The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids according to claim 1, characterized in that, The formation of hepatobiliary organoids in S1 includes the following stages: endoderm and mesoderm differentiation, hepatoblast induction, co-differentiation of hepatocytes and bile duct cells, and hepatocyte maturation. During the endoderm and mesoderm differentiation stage, the culture medium is based on the liver differentiation medium, with the addition of 2.5%-25% of stem cell culture medium mTeSR™ Plus and BMP4 differentiation factor 4-40 ng / mL to obtain hepatobiliary organoids containing immune cells.

8. The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids according to claim 7, characterized in that, The specific method for S1 is as follows: Phase 1: Endoderm and mesoderm are cultured in medium 1 to obtain endoderm and mesoderm. Medium 1 includes basic components and key additives. The basic components include RPMI1640 with a volume concentration of 2% B27-insulin-free. The key additives include mTeSR™ Plus with a volume concentration of 2.5%-25%, Activin A at 100-200 ng / mL, and BMP4 at 4-40 ng / mL. Phase 2: Hepatocytes were cultured in medium 2, which included basic components and key additives. The basic components included RPMI1640 with a volume concentration of 2% B27, and the key additives included mTeSR™Plus at a volume concentration of 2.5%-25%, FGF4 at 10-20 ng / mL, and BMP2 at 5-20 ng / mL. Phase 3: Hepatocytes and bile duct cells were cultured in culture medium 3, which included basic components and key additives. The basic components included RPMI1640 with a volume concentration of 2% B27, and the key additives included mTeSR™ Plus at a volume concentration of 2.5%-25%, HGF at 10-40 ng / mL, and FGF7 at 10-40 ng / mL. Phase 4: Culture in medium 4 until hepatocytes mature. Medium 4 includes basic components and key additives. The basic components include HCM medium, and the key additives include 5 ng / mL-20 ng / mL OSM and 0.1 μM-1.0 μM dexamethasone.

9. The method for constructing an HBV infection model based on multi-lineage hepatobiliary organoids according to claim 8, characterized in that, Culture in culture medium 1 for 1-4 days, in culture medium 2 for 3-5 days, in culture medium 3 for 5-7 days, and in culture medium 4 for 3-5 days.

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