Compositions and methods for long-term culture of hepatocytes

Abstract

Compositions for long-term maintenance of functional hepatocytes in culture, methods for improving maintenance of functional hepatocytes in vitro, and functional hepatocyte cultures according to the methods disclosed herein are provided. The compositions comprise at least: one adenylyl cyclase activator, one TGF inhibitor, one Notch inhibitor, one Wnt inhibitor, and / or one BMP inhibitor. The combination of compounds is added to any hepatocyte culture medium in an effective amount to long-term maintain functional hepatocyte function in vitro. The hepatocytes can be used for in vitro drug research and modeling of liver diseases.

Description

Technical Field

[0001] This invention mainly relates to the long-term maintenance and functional maturation of hepatocytes. Background Technology

[0002] Terminally differentiated cells are functionally stable in vivo, a state highly dependent on the precise spatiotemporal regulation of microenvironmental signals. Once isolated, cells undergo microenvironmental changes, leading to functional degradation. Maintaining terminally differentiated cells long-term in vitro has proven challenging. Once isolated, these cells typically lose their specific functions (Haenseler et al., StemCell Reports 8, 1727-1742 (2017); Lou et al., J Mol Cell Cardiol 51, 288-298 (2011); Ootani et al., Nat Med 15, 701-706 (2009); Shulman et al., Methods Mol Biol 945, 287-302 (2013). In particular, the inability to sustain primary human hepatocytes (PHH) for extended periods limits their application in modeling hepatotropic infection models such as the global pandemic of HBV infection (Zhong et al., Clin. Chim. Acta:412:1905-1911 (2011)). Advances such as the identification of the functional receptor for hepatitis B virus (HBV), Na+-taurocholate cotransport peptide (NTCP), have allowed for the establishment of hepatocellular carcinoma cell lines to study HBV infection (Yan et al., eLife). 1, (2012)). However, studying the HBV lifecycle over time, especially the persistence of cccDNA, remains challenging (Liang et al., Hepatology 62, 1893-1908 (2015)). Although PHH is considered ideal for HBV modeling, exhibiting highly similar infection characteristics to HBV infection from new humans, its long-term use is limited by limited in vitro viability and unstable functional maintenance (Lu et al., International journal of medical sciences 1, 21-33 (2004); Xia et al., J Hepatol 66, 494-503 (2017); Elaut et al., Curr Drug Metab 7, 629-660 (2006)). Therefore, establishing culture conditions for maintaining the function of truly human hepatocytes may be beneficial for HBV research.

[0003] Furthermore, the lack of effective culture conditions for stabilizing the function of terminally differentiated cells also limits the generation of functionally mature cells derived from human pluripotent stem cell (hPSC) differentiation and direct lineage reprogramming. Although hPSCs are considered an ideal potential cell source for generating a large number of diverse human cell types, most hPSC-derived cells exhibit an immature phenotype when generated using current protocols (Hrvatin et al., Proc Natl Acad Sci US A. 111(8) 3038-3043 (2014); Takayama et al., Drug Metab Pharmacokinet 32(1): 12-20 (2017)). One of the main reasons for the functional immaturity of hPSC-derived cells is the lack of available culture conditions that enable them to capture the final differentiation stage. Therefore, identifying culture conditions that support the maintenance of terminally differentiated cell function may be beneficial for their in vitro application and their generation.

[0004] Cell culture methods are still needed to provide solutions for the long-term functional maintenance of differentiated cell types, including human hepatocytes.

[0005] The purpose of this invention is to provide a method for long-term in vitro culture of functionally differentiated cells.

[0006] Another object of the present invention is to provide a composition for long-term maintenance of functionally differentiated cells in vitro.

[0007] Another object of the present invention is to provide differentiated cells with improved in vitro functional maintenance. Summary of the Invention

[0008] Compositions for long-term maintenance of functional hepatocytes in culture are provided, methods for improving the in vitro maintenance of functional hepatocytes, and functional hepatocyte cultures according to the methods disclosed herein.

[0009] The composition comprises at least: an activator of adenylate cyclase, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and / or a BMP inhibitor. In some preferred embodiments, the composition comprises a combination of an adenylate cyclase activator and a TGFβ inhibitor. In a particularly preferred embodiment, the composition comprises a combination of at least one adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and a BMP inhibitor (referred to herein as "5C"). The most preferred adenylate cyclase activator is fibrinogen (FSK); the preferred TGFβ inhibitor is SB431542 (SB43); the preferred Notch inhibitor is DAPT; the preferred Wnt inhibitor is IWP2; and the preferred BMP inhibitor is LDN193189 (LDN). The combination of compounds is added in an effective amount to any hepatocyte culture medium to maintain functional hepatocyte function in vitro for a long period.

[0010] The composition is used to improve the maintenance of functional hepatocytes in vitro.

[0011] Hepatocytes obtained according to the methods disclosed herein (“5C hepatocytes”) are also provided. When cultured according to the methods disclosed herein, the hepatocytes exhibit improved maintenance of hepatocyte function in vitro. In a preferred embodiment, after at least 60 days of culture, the 5C hepatocytes express at least six hepatocyte functional proteins at levels identical to those expressed by primary hepatocytes freshly isolated from the same organism. In a particularly preferred embodiment, after at least 90 days of culture, the 5C hepatocytes express at least six hepatocyte functional proteins at levels identical to those expressed by primary hepatocytes freshly isolated from the same organism. In a particularly preferred embodiment, exemplary hepatocyte functional proteins include albumin (ALB), APOA2, APOB, F2, F10, cytochrome P450 (CYP) 3A4, CYP1A2, CYP2C9, and UGT2B7.

[0012] A kit is provided for the long-term maintenance of functional hepatocytes in culture. The kit comprises an adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and a BMP inhibitor (“5C” herein). The most preferred adenylate cyclase activator is tuftin; the preferred TGFβ inhibitor is SB43; the preferred Notch inhibitor is DAPT; the preferred Wnt inhibitor is IWP2; and the preferred BMP inhibitor is LDN.

[0013] A method for identifying reagents that improve the maintenance of functionally differentiated cells, such as hepatocytes, in vitro is also disclosed. The method involves comparing the global gene expression profiles of freshly isolated primary hepatocytes with those cultured for only 24 hours, and screening for small molecule targets involved in hepatocyte fate patterns, differentiation, and reprogramming. Attached Figure Description

[0014] Figure 1 The mRNA levels of functional marker genes in hepatocytes cultured in HCM for 24 hours (24-h cultured hepatocytes) and freshly isolated hepatocytes (F-PHH) were shown by RNA-Seq analysis.

[0015] Figure 2A and 2B This demonstrates the identification of the TGF-β inhibitor (SB431542) as a factor that partially supports the maintenance of PHH in vitro. Figure 2A This study demonstrated the detection of genes enriched in the TGFβ signaling pathway in hepatocytes cultured in HCM for 24 hours (24-hour cultured hepatocytes) and freshly isolated hepatocytes (F-PHH) using RNA-Seq. Figure 2B Gene expression of mesenchymal marker genes and hepatocyte functional genes in PHH cultured under different conditions, as detected by qRT-PCR, is shown. n=2. Relative gene expression was normalized to F-PHH.

[0016] Figure 3A and 3B This study identified trichosine, a cAMP activator beneficial for in vitro PHH culture. A pool of candidate small molecules was screened to effectively inhibit EMT major gene expression and maintain the expression of key hepatocyte genes. qRT-PCR analysis was performed on day 14 of culture; F-PHH was used as a positive control. Relative gene expression was normalized to null values. n = 2.

[0017] Figures 4A-4C The combined effect of FSK and SB43 on PHH maintenance was demonstrated. Figure 4A This is a line graph showing the MTT assay used to analyze cell viability under different culture conditions. n=3. Figure 4B This study demonstrates the synergistic effect observed between SB43 and FSK (referred to as 2C) as detected by RT-qPCR on the regulation of mesenchymal marker gene and hepatocyte functional gene expression. Relative gene expression was normalized to F-PHH. Figure 4C The results show a qRT-PCR analysis of alternative liver function markers in cultured hepatocytes after 4 weeks of culture. Relative gene expression was normalized to F-PHH. n = 2. Figure 4D This is a line graph showing the ALB secretion kinetics in PHH cultured under different conditions. n = 3 independent batches. Figure 4EThis is a line graph showing albumin secretion and urea synthesis in cultured PHH. n=2. Figure 4F The drug metabolism activities of CYP enzymes in PHH cultured under different conditions are shown by LC / MS. Specific CYP3A4 activity was measured using testosterone (CYP3A4-T) and midazolam (CYP3A4-M). Results are expressed as pmol / min per million cells. n = 3. Figure 4G -I shows the results obtained via qRT-PCR ( Figure 4G ) and LC / MS ( Figure 4H Induction of CYP450 activity in 2C-PHH was detected. Rif (rifampin); PB (phenobarbital); ETOH (ethanol); BNF (β-naphthylflavonoid). n≥2. Figure 4I The inhibition of CYP450 activity in 2C-PHH cells was detected by LC / MS in 2C-cultured hepatocytes. n = 3. KC, ketoconazole; SX, sulfamethoxazole; ANF, α-naphthylflavonoid. Figure 4J The results are obtained by ELISA of human albumin in the serum of mice two and four weeks after transplantation of PHH cultured for 4 weeks. Figure 4K The expression of major CYP enzymes in PHH cultured for 14 and / or 30 days at 2C conditions was shown by qRT-PCR. Two basal media were used: William E (WME) and HCM. Relative gene expression was normalized to F-PHH.

[0018] Figures 5A-5E The identification of the optimal configuration (5C conditions) for maintaining PHH is shown. Figure 5A This is a protocol for demonstrating chemical screening to discover candidates that synergize with SB43 and FSK. Figure 5B The effects of combining DAPT, LDN193189, and IWP2 with SB43 and FSK, respectively, on EMT gene expression (suppression) or liver function gene expression (enhancement) were shown. Relative gene expression was normalized to DMSO. n = 2. Figure 5C The effects of three additional small molecules (IWP2, DAPT, and LDN193189) were demonstrated; when used in combination with SB43 and FSK, they reduced the expression of the PHH intermediate mesenchymal marker gene COL1A1 in cultured cells. qRT-PCR analysis was performed on day 14. Relative gene expression was normalized to FSK+SB43. n=3. Figure 5D This demonstrates the role of small molecules or cytokines targeting the same signaling pathway in inhibiting EMT markers, replacing IWP2, DAPT, and LDN193189. Relative gene expression was normalized to FSK+SB43. n=3. Figure 5EThe effect of each 5C small molecule on maintaining PHH was demonstrated using a single small molecule omission assay. The expression of liver transcription factors and functional genes was detected by qRT-PCR. Relative gene expression was normalized to 5C. n=2.

[0019] Figure 6A qRT-PCR analysis of mesenchymal marker genes in frozen PHH cells cultured for 2 weeks in hepatocyte medium (empty) and 5C culture conditions (5C) is shown. DMSO was used as an excipient control. n=3. Similar results were obtained in 3 independent batches. Figure 6B and 6C Showing freshly isolated PHH from Asian donors ( Figure 6B ) and cryopreserved PHH recovered from Caucasian donors ( Figure 6C Time-course analysis of human albumin secretion and urea synthesis maintained at 5C and control conditions. n=3. Figure 6D and 6E The study showed that 5C supports PHH maintenance for more than 2 months. Figure 6D The study showed the albumin secretion and urea synthesis of PHH cultured at 5C for 60 days. n=3. Similar results were obtained in two independent batches. Figure 6E qRT-PCR analysis of key liver genes in hepatocytes cultured at 5C (5C-PHH) on days 60 and 90. Relative expression levels were normalized to F-PHH. n = 2.

[0020] Figure 7 Hierarchical clustering was shown to compare the global gene expression profiles of PHH cultured under different conditions, hepatocytes derived from pluripotent stem cells (hiPSC-Hep_rep1 / 2 / 3 / 4), freshly isolated human hepatocytes (PHH 0d), and adult liver tissue. Three independent batches of PHH were applied, with corresponding adult liver tissues used as positive controls. Expression profiles of PHH 0d and hiPSC-Hep_rep1 / 2 / 3 / 4 were obtained from RNA-seq data obtained by Gao et al., Stem Cell Reports 9, 1813-1824 (2017).

[0021] Figure 8A Gene expression analysis of liver replacement function markers and mesenchymal markers in PHH maintained in 5C-PHH and sandwich culture conditions was performed by qRT-PCR. qRT-PCR analysis was conducted on day 15. Relative expression levels were normalized to F-PHH. n=2. Figure 8B Gene expression analysis of liver transcription factors by qRT-PCR is shown. Relative expression levels were normalized to F-PHH. n=2. Similar results were obtained in two independent batches. Figure 8CThe effect of 5C on the drug metabolism activity of cultured human hepatocytes was shown by analyzing the gene expression of five core CYP450 enzymes by qRT-PCR after 4 weeks of culture. n=3. Figure 8D The qRT-PCR analysis of the major phase II enzymes and phase III transporters in 5C-PHH after 4 weeks of culture is shown. Relative expression levels were normalized to F-PHH. n=2. Similar results were obtained in at least 3 independent batches. Figure 8E Dynamic gene expression analysis of drug metabolism-related genes in frozen PHH under different conditions was presented by qRT-PCR. After 1 week of culture under control conditions (empty and DMSO), recovered frozen PHH showed severe cell death, therefore no data were available after day 10. Relative gene expression was normalized to frozen PHH. n = 2. Figure 8F The effect of 5C on the drug metabolism activity of cultured human hepatocytes was shown. The drug metabolism activity of five core CYP450 enzymes was measured by LC-MS after 2 weeks of culture. n=3. Data not detected were marked as "nd".

[0022] Figure 9A -F indicates that 5C supports the entire lifecycle of HBV infection in PHH. Figure 9A Gene expression analysis of NTCP was performed using qRT-PCR. n=3. Figure 9B The kinetics of HBV antigen (HBsAg and HBeAg) formation in the supernatant of infected PHH cultured under different conditions. n=3. Figure 9C The kinetics of HBV RNA formation in PHH cultured under different conditions were analyzed by qRT-PCR. n=2. Figure 9D : Kinetics of HBV DNA formation in PHH supernatant cultured under different conditions. n=3. Figure 9E : Kinetics of cccDNA formation in 5C-PHH as detected by qPCR. n=2. Figure 9F and 9G HBeAg (n=3) was observed in PHH inoculated with HBV-infected 5C-PHH supernatant. Figure 9F ) and viral DNA (n=2) Figure 9G ) detection. Figure 9HThis is a Southern Blot analysis of HBV cccDNA from infected 5C-PHH cells. HBV DNA was extracted using a core DNA extraction method (lanes 1 and 4) and a modified Hirt DNA extraction method (lanes 2, 3, 5, and 6). To confirm cccDNA identity, untreated (lanes 2 and 5) and SpeI-digested (lanes 3 and 6) Hirt DNA preparations extracted from 5C-PHH were separated on agarose gels. Dpi represents days post-infection.

[0023] Figure 10A -B indicates HBV infection in 5C-PHH with a decreased multiple of infection (MOI). This is achieved through the secretion of HBV antigens (HBsAg and HBeAg). Figure 10A ) and intracellular HBV transcripts ( Figure 10B ) Detection of HBV infection. n=3.

[0024] Figure 11A The images show HBsAg and HBeA in PHH samples isolated from Caucasian and Asian donors infected with HBV type B or C and further cultured at 5°C. n = 3. Figure 11B -D indicates the effect of 5C-PHH infection with HBV particles generated from HepAD38. Figure 11B Time-history analysis of HBsAg and HBeAg levels in the supernatant of 5C-PHH. n=3. Detected at 7 dpi and 18 dpi. Figure 11C HBV RNA, ( Figure 11D HBV intracellular DNA and cccDNA. n=3. Similar results were obtained in at least 3 independent batches. Figure 11E -F indicates that the neutralizing antibody (anti-S antibody) blocked the spread of HBV in 5C-PHH. Figure 11E Time-course analysis of HBeAg in 5C-PHH infected with different MOIs. Neutralizing antibody (anti-S antibody) was added from 1 Dpi. n = 3. Figure 11F Quantification of HBcAg-positive cells at Dpi 28. n=3.

[0025] Figure 12A -D shows the response of 5C-PHH in HBV infection to anti-HBV drugs. Figure 12A -D shows the effect of anti-HBV drugs (ETV, LAM, and IFN-α) on HBV antigens (HBsAg and HBeAg) in infected 5C-PHH ( Figure 12A ), HBV RNA (detected at 16 Dpi) Figure 12B ), HBV DNA (detected at 21 dpi) Figure 12C) and HBV cccDNA (detected at 34 dpi) Figure 12D The role of ).

[0026] Figure 13A This is a time-course analysis of the inflammatory response in cultured 5C-PHH. Relative gene expression was normalized to 5C. n=3. Similar results were obtained in 3 independent batches. Figure 13B -D is a comparison of 5C with three other reported PHH culture conditions in terms of long-term hepatocyte maintenance and HBV permissibility. Condition 1, C1 (Winer et al., Nat Comm. 8:125 (2017)); Condition 2, C2 (Lucifora et al., Science, 343:1221-28 (2014)); and Condition 3, C3 (Xia et al., J. Hepatol., 66:494-503 (2017)). Figure 13B qRT-PCR analysis of liver function gene expression in PHH cultured under different culture conditions. n=3. Figure 13C Time-course analysis of human albumin secretion and urea synthesis in PHH cultured under different culture conditions. n=3. Figure 13D Time-course analysis of HBeAg. PHH was infected with plasma from hepatitis B patients and further cultured for extended periods under different conditions. n=3. Similar results were obtained in 3 independent batches.

[0027] Figure 14A -C shows the application of 5C culture conditions to various plate formats. PHH was inoculated into microplates (96-well and 384-well plates) and further cultured in 5C. Figure 14A EMT markers and liver function genes were analyzed after 2 weeks of culture. Figure 14B ALB secretion and urea synthesis were measured after 7 and 14 days of culture. Figure 14C HBV infection was performed in microplates, and HBsAg and HBeAg were analyzed. n=3. Similar results were obtained in two independent batches.

[0028] Figure 15A -C indicates the effect of 5C on HCV infection in frozen PHH. Frozen PHH were infected with HCVcc (MOI=2) and then further cultured in 5C with or without DCV treatment. Figure 15A B) measured the secreted HCV products ( Figure 15A ) and intracellular HCV RNA ( Figure 15B n = 3. Figure 15C The qRT-PCR analysis of ISG expression is shown in HCV-infected (shown here as Jc1G) and uninfected 5C-PHH. n=3.

[0029] Figure 16 The expression kinetics of a group of CYP enzymes, drug-metabolizing nuclear receptors, and key transcription factors in human embryonic stem cell-derived hepatocytes (hESC-Hep) cultured under 2C conditions were demonstrated. Relative gene expression was detected by qRT-PCR and normalized to day 0. n=2. Invention Details

[0031] In this paper, we demonstrate that this fundamental problem in primary human hepatocytes (PHHs) can be overcome by modulating cell signaling pathways using a combination of small molecules. Five chemical conditions (5Cs) for the long-term functional maintenance of PHHs were identified. PHHs cultured in 5Cs exhibited a global gene expression profile and hepatocyte-specific functions similar to those of freshly isolated human hepatocytes. PHHs cultured in 5Cs allowed for hepatitis B virus (HBV) infection and effectively reenacted the entire HBV infection process within 4 weeks, maintaining persistently detectable HBV covalently closed circular DNA (cccDNA). Our study provides an efficient platform for understanding HBV pathology and screening antiviral drugs, and this chemical approach can be applied to the maintenance of other functional cell types in vitro.

[0032] The disclosed culture conditions also maintain and promote near-adult-stage hESC-hepatocytes. Under our conditions, hESC-hepatocytes can be maintained for more than one month, and functional maturation of long-term cultured hESC-hepatocytes was observed. hESC-hepatocytes cultured under our conditions gradually expressed a group of CYP enzymes, indicating that hESC-hepatocytes are approaching an adult-like stage of functional maturation, which facilitates the generation of functionally mature cells from hPSCs.

[0033] I. Definition

[0034] As used herein, “5C medium” refers to a basic hepatocyte culture medium supplemented with at least one adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and a BMP inhibitor, such as HCM (hepatocyte culture medium, Lonza) or William's E medium containing 2% B27 (Gibco), 1% GlutaMAX, and supplemented with saliva extract, SB431542, DAPT, IWP2, and LDN193189.

[0035] As used herein, “culture” means a population of cells that is grown in a culture medium and optionally passaged. A cell culture may be a primary culture (e.g., a culture that has not yet been passaged) or a secondary or subsequent culture (e.g., a population of cells that has been subcultured or passaged once or multiple times).

[0036] As used in this article, “downregulation” or “lower regulation” refers to the process by which cells reduce the amount and / or activity of cellular components such as DNA, RNA, or proteins in response to external variables.

[0037] As used in this article, the term "induced hepatocytes" (iHep) refers to cells that are not naturally occurring hepatocytes but are artificially derived from non-hepatocytes.

[0038] The terms “oligonucleotide” and “polynucleotide” generally refer to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, as used herein, a polynucleotide refers to single-stranded and double-stranded DNA, a mixture of single-stranded and double-stranded regions of DNA, single-stranded and double-stranded RNA, and a mixture of single-stranded and double-stranded regions of RNA, a hybrid molecule containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. The terms “nucleic acid” or “nucleic acid sequence” also encompass polynucleotides as defined above.

[0039] As used herein, the term polynucleotide includes the aforementioned DNA or RNA containing one or more modified bases. Therefore, DNA or RNA with a backbone modified for stability or other reasons is a "polynucleotide" as the term refers herein. Furthermore, to name just two examples, DNA or RNA containing unusual bases (e.g., inosine) or modified bases (e.g., triphenylmethylated bases) is a polynucleotide as used herein.

[0040] As used in this article, "reprogramming" refers to transforming one cell type into another. For example, cells that are not hepatocytes can be reprogrammed into cells that are morphologically and functionally similar to hepatocytes.

[0041] The term “upregulation of expression” refers to influencing expression, such as inducing expression or activity, or inducing increased / greater expression or activity relative to untreated cells.

[0042] As used in this article, “upregulation” or “upmodulation” refers to the process by which cells increase the amount and / or activity of cellular components such as DNA, RNA, or proteins in response to external variables.

[0043] II. Composition

[0044] The composition includes hepatocyte culture medium supplemented with at least: an adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and / or a BMP inhibitor.

[0045] In some preferred embodiments, the composition comprises a combination of an adenylate cyclase activator and a TGFβ inhibitor (2C herein).

[0046] In a more preferred embodiment, the composition comprises a combination of an adenylate cyclase activator and a TGFβ inhibitor with at least one agent selected from the group consisting of Notch inhibitors, Wnt inhibitors and BMP inhibitors.

[0047] In a particularly preferred embodiment, the composition comprises a combination of at least one adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and a BMP inhibitor (referred to herein as "5C"). The most preferred adenylate cyclase activator is laryngin; the preferred TGFβ inhibitor is SB43; the preferred Notch inhibitor is DAPT; the preferred Wnt inhibitor is IWP2; and the preferred BMP inhibitor is LDN193189.

[0048] A combination of compounds is added in an effective amount to any hepatocyte culture medium to maintain functional hepatocyte function in vitro for an extended period, as measured in some embodiments as follows: an effective amount of the compounds that maintains the expression of at least six functional hepatocyte proteins at the same level as freshly isolated primary hepatocytes from the same organism after at least 60 days of culture.

[0049] Useful cell cultures for hepatocyte culture are known in the art (hepatocyte basal culture media herein). Useful examples include, but are not limited to, Williams Medium E containing B27, Glutmax, and penicillin-streptomycin; hepatocyte culture media; Dulbecco Modified Eagle Medium (“DMEM”) and Ham’s F12 Medium, used alone or in combination; Waymouth’s MB-752 / 1, Ham’s F12, RPMI 1640, Dulbecco Modified Eagle Medium, Leibovitz L15, and HBM. TM (Lonza), hepatocyte basal medium WAJC 110 (MyBiosouirce.com), and modified Chee's medium.

[0050] A. Liver cell culture medium supplement

[0051] cAMP agonists

[0052] Preferred cAMP agonists are trichodin, used at concentrations ranging from 2 to 100 μM, preferably 5 to 50 μM, and more preferably 18 to 22 μM. However, the CINP mixtures disclosed herein may include any cAMP agonist. Examples include, but are not limited to, prostaglandin E2 (PGE2), leuprolide, genistein, and cAMP analogs such as DBcAMP or 8-bromo-cAMP.

[0053] TGFβ inhibitors

[0054] In some embodiments, the TGFβ inhibitor preferably inhibits TGFβ1 receptor-activated receptor-like kinase (ALK)5, and in other embodiments, it may additionally inhibit ALK4 and nodular receptor 1 receptor ALK7. The TGFβ receptor inhibitor may be SB431542 (4-[4-(1,3-benzodioxazol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide), the structure of which is shown below, used at concentrations of 2-50 μM, preferably 2-15 μM, more preferably 8-12 μM.

[0055]

[0056] Other TGFβ inhibitors known in the art and commercially available. Examples include E-616452 ([2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthidine]; 83-01 [3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazol-1-thiocarbonylamide (carbothioamide)]; SB505124 [2-[4-(1,3-benzodioxazol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine]; GW 788388 [4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide]; and SB 525334[6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridyl)-1H-imidazol-4-yl]quinoxaline] and dosomorphine.

[0057] Notch inhibitors

[0058] The composition preferably contains one or more notch inhibitors, more preferably DAPT ((2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethyl ethyl ester), the structure of which is shown below, and is used at a concentration of 0.1-10 μM, preferably 0.5-2.5 μM, more preferably 1-2 μM.

[0059]

[0060] Other notch inhibitors are known in the art. Representative examples include MK 0752, whose structure is shown below, used at concentrations of 0.02-20 μM, preferably 0.2-10 μM, and more preferably 1-5 μM.

[0061]

[0062] RO4929097, the structure of which is shown below, is used at a concentration of 0.02-20 μM, preferably 0.2-10 μM, and more preferably 1-5 μM.

[0063]

[0064] XXi(S,S)-2-[2-(3,5-difluorophenyl)-acetamido]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide), preferably used at a concentration of 0.2-1 μM; DBZ(N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenzo[b,d]diazepin-7-yl]amino]-1-methyl- 2-Oxyethyl]-3,5-difluorophenylacetamide); SAHM1 (peptide sequence AERLRRRIXLCRXHHST with the following modifications: Ala-1 = N-terminal Ac, Ala-1 = β-Ala, X = (S)-2-(4-pentenyl)alanine, X-9 and X-13 linked together by a double bond); and FLI06 (cyclohexyl 1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-3-quinoline carboxylate).

[0065] Wnt inhibitors

[0066] The disclosed compositions may contain one or more Wnt inhibitors. A preferred Wnt inhibitor is IWP2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthiophene[3,2-d]pyrimidin-2-yl)[thio]acetamide), the structure of which is shown below, and is used at concentrations of, for example, 0.01-2.5 μM, preferably 0.1-1 μM, and more preferably 0.5-1 μM.

[0067]

[0068] IWP2 inhibits Wnt processing and secretion. It can inactivate PORCN, a membrane-bound O-acyltransferase (MBOAT), and selectively inhibit Wnt palmitoylation. It blocks Wnt-dependent phosphorylation of the Lrp6 receptor and Dvl2, as well as the accumulation of β-catenin.

[0069] Other Wnt signaling inhibitors are known in the art. Representative examples include Dickkopf-associated protein 1 (DKK1), preferably used at a concentration of 10-1000 ng / ml, more preferably 50-150 ng / ml; IWR1 (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methanol-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide), preferably used at a concentration of 0.1-5 μM, more preferably 1-2 μM; Wnt-C59 (4-(2-methyl-4-pyridyl)-N-[4-(3-pyridyl)phenyl]phenylacetamide), preferably used at a concentration of 0.01-5 μM, more preferably 0.05-1 μM; IWP L6(N-(5-phenyl-2-pyridyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthiophene[3,2-d]pyrimidin-2-yl)thio]acetamide) and IWP 12(N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3,6-dimethyl-4-oxothiaphene[3,2-d]pyrimidin)-2-yl)thio]acetamide).

[0070] BMP inhibitors

[0071] The composition comprises one or more BMP inhibitors. A preferred BMP inhibitor is LDN193189 2HCl (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride), the structure of which is shown below, and is used at concentrations of, for example, 0.01-0.5 μM, preferably 0.05-0.25 μM, more preferably 0.1-0.2 μM.

[0072]

[0073] LDN193189 is a potent and selective ALK2 and ALK3 inhibitor (ICV). 50 The values ​​were 5 and 30 nM, respectively; it inhibited BMP4-mediated Smad1 / 5 / 8 activation. The selectivity of BMP signaling was more than 200 times greater than that of TGF-β signaling. It also showed selectivity for AMPK, PDGFR and MAPK signaling.

[0074] Other useful BMP inhibitors are known in the art. Representative examples include, but are not limited to, LDN 212854 (5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline); the structure of which is shown below, and is preferably used at a concentration of 0.05-5 μM, more preferably 0.1-2 μM.

[0075]

[0076] Noggin, preferably used at a concentration of 10-1000 ng / ml, more preferably 50-500 ng / ml; Dorsomorphin dihydrochloride (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride); and K02288 (3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol).

[0077] B. Hepatocytes cultured in a small molecule supplemented medium

[0078] Hepatocytes cultured using the supplemented hepatocyte culture medium disclosed herein are used to maintain functional hepatocytes in vitro for extended periods, as measured by hepatocytes possessing at least one of the following characteristics: hepatocyte morphology (polygonal shape), established hepatocyte functions such as albumin secretion and urea synthesis, and expression of at least one known hepatocyte marker. The expression level of the hepatocyte marker can be determined by measuring the expression level of the oligonucleotide encoding the hepatocyte marker or by measuring the protein level using methods known in the art and illustrated herein.

[0079] In one embodiment, the ability to maintain functional hepatocytes in vitro for a long period is measured based on hepatocytes possessing at least one of the listed characteristics after at least two weeks of culture. In some embodiments, hepatocytes possess at least one of the listed characteristics after three weeks of culture. For example, hepatocytes possessing at least one of the listed characteristics after 4, 5, 6, 7, 8, 9 weeks, and up to 90 days or longer of culture, and their levels are improved when compared to hepatocytes obtained from the same organism and cultured without supplementing the cell culture medium with the hepatocyte cell culture supplement disclosed herein.

[0080] Hepatocyte markers that can be used to determine the long-term maintenance of functional hepatocytes in cultures include, but are not limited to, cytochrome P450 (CYP) 3A4; CYP1A2; CYP2C9; CYP2D6; CYP2B6; CYP2C8; CSP1; CYP2C19; UDP-glucuronyltransferase (UGT) 1A3; UGT1A4; UGT2B15; UGT2B7; NTCP (Na+-taurocholate cotransporter); ABCG2 (ATP-binding cassette superfamily G member 2, also known as BCRP); MRP2 (multidrug resistance-associated protein 2); organic anion transporter (OATP) 1B1; APOA2; APOB; F2 (coagulation factor II); and F10 (coagulation factor X). Therefore, hepatocytes cultured in the supplemented hepatocyte culture medium disclosed herein showed improved expression of at least one of P450(CYP)3A4; CYP1A2; CYP2C9; CYP2D6; CYP2B6; and CYP2C19 compared to hepatocytes from the same organism cultured in the same basal hepatocyte culture medium but without supplementation of the disclosed small molecules. The improved expression was measurable as a statistically significant difference in expression levels.

[0081] In some preferred embodiments, hepatocytes cultured according to the methods disclosed herein express at least one of the listed hepatocyte markers, and their levels are comparable (measured as not statistically significant) or higher than those of hepatocytes freshly isolated from the same organism.

[0082] In a particularly preferred embodiment, hepatocytes maintained in vitro using the supplementary cell culture medium disclosed herein express at least one, at least two, at least three, at least four, at least five, or at least six of the following hepatocyte markers: APOA2, APOB, F2, F10, CYP3A4, CYP1A2, CYP2C9, and UTG2B7, at levels comparable to those of freshly isolated hepatocytes obtained from the same organism after 90 days of culture.

[0083] Hepatocytes cultured in 5C medium showed metabolically active CYP3A4, CYP1A2, CYP2C9, CYP2D6, and CYP2B6 levels after 2 weeks of culture, comparable to those in F-PHH. In contrast, the metabolic activity of PHH cultured in the control group (hepatocyte medium without 5C supplementation) was almost undetectable at the same time point.

[0084] III. How to Use

[0085] A. Hepatocyte culture

[0086] The disclosed cell culture composition is used for hepatocyte culture to maintain functional hepatocytes in vitro for a long period. The disclosed cell culture composition can be used not only to maintain hepatocytes but also for the functional maturation of hepatocytes (e.g., hepatocytes generated through differentiation or reprogramming).

[0087] In some embodiments, the disclosed method does not rely on the co-culture of hepatocytes with a second cell type, such as stellate cells. However, other embodiments may use co-culture formats that include a second cell type, such as stellate cells. In some embodiments, the cell culture is a sandwich culture format, as disclosed, for example, in Dunn et al., Biotechnol. Prog., 7:237-245 (1991).

[0088] Hepatocytes from any source can be cultured using the supplementary cell culture media disclosed herein. Hepatocytes can be isolated from animals such as mice, livestock, primates, or humans. In a preferred embodiment, the hepatocytes are isolated from humans. The hepatocytes can also be induced hepatocytes (iHep). For example, iHep can be obtained through reprogramming or differentiation. In some preferred embodiments, the hepatocytes are primary hepatocytes, and in some embodiments, the hepatocytes can be cryopreserved hepatocytes.

[0089] Primary hepatocytes can be isolated from donors using methods known in the art, such as a two-step collagenase perfusion procedure (Lee et al., J Vis Exp, 79, e50615 (2013)). For example, liver samples are perfused with a suitable perfusion buffer (PB) to remove blood cells, followed by treatment with perfusion buffer plus EDTA (PBE) and subsequently perfusion buffer plus collagenase (PBC). An exemplary perfusion buffer is 0.15 M NaCl; 5 mM KCl; 25 mM NaHCO3; 5 mM glucose; 20 mM Hepes). The hepatocyte suspension is washed with a hepatocyte basal medium, such as Williams medium E, filtered, and the isolated hepatocytes are seeded in tissue culture plates / matrix for hepatocyte culture and cultured in a basal hepatocyte medium supplemented with one adenylate cyclase activator, one TGFβ inhibitor, one Notch inhibitor, one Wnt inhibitor, and / or one BMP inhibitor.

[0090] In some implementations, isolated hepatocytes are seeded onto a monolayer of collagen, with or without a second layer of collagen added to provide a sandwich-type hepatocyte culture. Hepatocytes can also be cultured into a form resembling hepatocyte spheroids when cultured on a soft or non-adhesive extracellular matrix.

[0091] Preferred culture conditions include a humidified incubator at 37°C and 5% CO2.

[0092] The basal hepatocyte culture medium is supplemented with at least one of adenylate cyclase, TGFβ inhibitor, Notch inhibitor, Wnt inhibitor, and BMP inhibitor. In some preferred embodiments, the basal hepatocyte culture medium is supplemented with a combination of adenylate cyclase activator and TGFβ inhibitor.

[0093] In a more preferred embodiment, the basal hepatocyte culture medium is supplemented with an adenylate cyclase activator and a TGFβ inhibitor, along with at least one reagent selected from the group consisting of Notch inhibitors, Wnt inhibitors, and BMP inhibitors.

[0094] In a particularly preferred embodiment, the basal hepatocyte culture medium is supplemented with a combination of at least one adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and a BMP inhibitor (hereinafter referred to as "5C"). The most preferred adenylate cyclase activator is tuftin; the preferred TGFβ inhibitor is SB431542 (SB43); the preferred Notch inhibitor is DAPT; the preferred Wnt inhibitor is IWP2; and the preferred BMP inhibitor is LDN193189.

[0095] B. Research related applications

[0096] The research disclosed in this paper shows that human hepatocytes cultured according to the methods disclosed in this paper can maintain drug metabolic activity for a long time, thereby providing a platform for drug application.

[0097] (i) Drug testing

[0098] Hepatocytes play a crucial role in drug development because the liver is central to drug metabolism. Currently, a major reason for candidate drug failure is its suboptimal ADME (absorption, distribution, metabolism, and excretion). A significant part of drug discovery research involves the metabolic and toxicological effects of candidate drugs on hepatocytes, and human hepatocytes are fully involved in drug metabolism. Currently, the main hepatocytes used for in vitro drug development are adult primary hepatocytes. However, their application in drug development is very limited due to the difficulty in maintaining the function of primary hepatocytes in vitro. Hepatocytes cultured according to the method disclosed in this paper express drug metabolism genes for extended periods in culture and maintain metabolic activity for a long time, making them suitable for in vitro drug metabolism studies. As mentioned above, hepatocytes cultured in 5C showed metabolically active CYP3A4, CYP1A2, CYP2C9, CYP2D6, and CYP2B6 levels after 2 weeks of culture, comparable to those of F-PHH. In contrast, the metabolic activity of PHH cultured in the control group (hepatocyte culture medium without 5C supplementation) was almost undetectable at the same time point.

[0099] (ii) Liver disease model

[0100] A problem encountered in research involving infectious diseases is the lack of suitable models. Cultured hepatocytes can be used as cellular models for studying infections caused by liver parasites, such as hepatitis viruses (hepatitis B, C, or D) and infections caused by parasites that cause malaria. These models can provide an effective platform for developing vaccines and drugs to treat infectious diseases, particularly those affecting the liver.

[0101] Hepatocytes cultured according to the methods disclosed herein can be used as in vitro models to study liver diseases, such as viral infections of the liver. Preferred examples are hepatitis virus infection and disease progression. In a particularly preferred embodiment, hepatocytes can be used as models of hepatitis B and hepatitis C infection and disease progression.

[0102] Therefore, the hepatocytes disclosed herein can be used as an in vitro model of liver parasitic infection, which is generated by a method comprising: inoculating the hepatocytes of the present invention into a liver parasitic organism for a period of time during which the infection is effective, and culturing the infected cells in vitro in a liver basal medium supplemented with HC.

[0103] Data in this application demonstrate that inoculation of PHH (cultured according to the methods disclosed herein) with HBV particles generated from plasma or cell cultures of HBV-infected patients resulted in successful HBV infection, with HBV products (secreted HBsAg, HBeAg, and DNA particles) detected in the supernatant of 5C-PHH cultures. High levels of HBV product generation persisted in 5C-PHH for at least one month. HBV cccDNA formation was detectable after 4 weeks of culture under 5C conditions. Effective HBV infection by HBV core antigen (HBcAg) immunostaining was demonstrated by 5C-PHH (data not shown). Furthermore, 5C-PHH remained susceptible to HBV infection even when the MOI (multiple of infection) decreased to 10. HBV infection was achieved at MOI = 10, indicating that 5C-PHH is a highly susceptible system for HBV infection.

[0104] Importantly, 5C can stably support HBV infection from various sources, including various HBV genotypes and the in vitro production cell line HepAD38. 5C-PHH can support the entire HBV life cycle and produce HBV infectious particles.

[0105] In addition, 5C-PHH can also be infected with other hepatitis viruses, such as hepatitis C virus (HCV).

[0106] Hepatocytes cultured according to the methods disclosed in this article can also be used to describe treatment options / efficacy of new therapeutic agents for liver diseases.

[0107] The data in this application illustrate hepatocytes cultured according to the information disclosed herein that respond to viral therapy. The 5C-PHH infection model is sensitive to two classes of clinical anti-HBV drugs.

[0108] Successful inoculation of hepatocytes with hepatitis viruses can be extended to infecting other parasites of the liver, such as malaria parasites.

[0109] Malaria is a mosquito-borne infectious disease caused by microorganisms belonging to the Plasmodium family; it affects humans and other animals. Five species of Plasmodium can infect and be transmitted from human to human. Most deaths are caused by *Plasmodium falciparum*, as *Plasmodium vivax*, *Plasmodium ovale*, and *Plasmodium malariae* typically cause milder forms of malaria. The disease is most commonly transmitted by infected female *Anopheles* mosquitoes. A mosquito bite introduces the parasite, in the mosquito's saliva, into the bloodstream of the bitten individual. The parasite enters the liver, where it matures and multiplies.

[0110] V. Reagent Kit

[0111] A kit is provided for the long-term maintenance of functional hepatocytes in culture. The kit comprises an adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor, and a BMP inhibitor (referred to herein as "5C"). The most preferred adenylate cyclase activator is tuftin; the preferred TGFβ inhibitor is SB431542 (SB43); the preferred Notch inhibitor is DAPT; the preferred Wnt inhibitor is IWP2; and the preferred BMP inhibitor is LDN193189. The kit includes instructions for supplementing basal hepatocyte culture medium with at least one adenylate cyclase activator, one TGFβ inhibitor, one Notch inhibitor, one Wnt inhibitor, and one BMP inhibitor. Example

[0112] Materials and methods

[0113] Human hepatocyte isolation and culture

[0114] This study was approved by the Research Ethics Committee of the China-Japan Friendship Hospital (Ethics Approval No.: 2009-50). PHH cells were obtained from human donors. Human hepatocytes were isolated from discarded human livers. Primary human hepatocytes were isolated from these tissues using a modified two-step collagenase perfusion procedure (OOtani et al., Nat Med., 15:701-706 (2009)). Briefly, liver tissue was perfused with PB (perfusion buffer: 0.15M NaCl; 5mM KCl; 25mM NaHCO3; 5mM glucose; 20mM Hepes) to remove residual blood cells, followed by PBE (perfusion buffer plus 1mM EDTA). The tissue was then perfused with PBC (perfusion buffer plus 1 mg / mL type IV collagenase, 5mM CaCl2, Gibco). All buffers were preheated to 37°C prior to the separation process. Hepatocyte suspension was collected and washed with Williams E (Gibco) medium, then filtered through a 40 μm nylon cell filter (Falcon). The isolated hepatocytes were then separated at 2.5 x 10⁻⁶ mm. 5 Inoculated at a density of / cm2 on type I collagen-coated (rat tail collagen type I, Gibco) plates.

[0115] Commercial hepatocytes: Plated and cryopreserved human hepatocytes (lots: HVN and HNN) were purchased from Bioreclamation IVT (product number: M00995-P).

[0116] Serum-free medium (Null) for hepatocyte culture: Williams medium E containing B27 (50x, Gibco), Glutmax (Gibco), and penicillin-streptomycin (Pen Strep) (Gibco). DMSO excipient control: Null, supplemented with approximately 0.035% DMSO. 5C conditions: Null, supplemented with trichodin (10 μM), SB431542 (10 μM), IWP 2 (1 μM), DAPT (1 μM), and LDN193189 (0.5 μM). Hepatocytes cultured for 24 hours in commercial HCM (Lonza) medium.

[0117] Cryopreservation and recovery of human liver cells

[0118] hepatocytes at 1*10 7Cells / mL (1 mL / vial) were cryopreserved in a medium containing 60% Williams E (Gibco), 30% HypoThermosol FRS (BioLife Solutions), and 10% DMSO (Sigma-Aldrich). The vials were then immediately transferred to a Cryo 1°C freezer (Nalgene) and frozen at -80°C for 24 hours. Subsequently, the vials were transferred to liquid nitrogen for long-term storage.

[0119] For recovery, the cryopreserved hepatocyte vials were thawed in a 37°C water bath. Then, 1 mL of cell suspension was added to 5 mL of Null, centrifuged at 160 g for 5 minutes at 4°C, and resuspended in culture medium. Cell viability was verified using trypan blue (Gibco).

[0120] Small molecule compounds and cytokines

[0121] The small molecule compounds and cytokines used for screening were purchased or synthesized, as described in Table 1. Their concentrations are shown in Table 1.

[0122] Table 1. Small molecules or cytokines used in hepatocyte culture.

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Drug metabolic activity assay

[0130] Drug metabolic activity was assessed using the previously described method (Kyffin et al., Toxicol. In vitro. An Int. J. Published inassoc with BBIBRA, 48:262-275 (2018)). Briefly, hepatocytes were collected as a cell suspension to assess drug metabolic activity. 1 mL of pre-warmed incubation medium (Williams E, 10 mM Hepes, pH 7.4, GlutaMAX) was added to 1 x 10⁻⁶ cells / mL of the suspension. 6In cells. Substrate solutions were prepared by adding the substrates individually to the incubation medium. The metabolic reaction was initiated by mixing 250 μl of substrate solution with 250 μl of cell suspension in 5 mL polystyrene round-bottom tubes (BD Falcon). The tubes were placed on an orbital vibrator in an incubator set to a vibration speed of 210 rpm. After incubation at 37 °C for 15–120 min, the reaction tubes were centrifuged at room temperature, and the supernatant was collected. The drug metabolism reaction was stopped by adding three volumes of methanol to the collected supernatant and freezing at -80 °C. To quantify the metabolites, 1% internal control was added for each substrate, and liquid chromatography / mass spectrometry (LC / MS) analysis was performed. The results were normalized to pmol of metabolite formed per million cells per minute. Specific substrates and their concentrations for each CYP enzyme are shown in Table 2. Isotope-labeled reference and standard metabolites are listed in Table 2.

[0131] Table 2. CYP enzyme-selective substrates used in HPLC.

[0132]

[0133] Preparation of hepatitis B virus stock solution

[0134] HBV virus was obtained from the plasma of chronic HBV carriers with written consent. HepAD38 cells were cultured in Dulbecco modified minimum essential medium (DMEM) supplemented with 10% FBS and 0.1 mM non-essential amino acids (NEAA). After three days, when the cells reached confluence, the medium was replaced with a medium consisting of 3% FBS, 0.1 mM NEAA, and 2% DMSO. The supernatant was collected every three days and fresh medium was added. The collected medium was combined and concentrated 100-fold by centrifugation using an Amicon Ultra-15 centrifuge (Regenerated Cellulose 100,000, Merck Millipore Corp.). The concentrated viral stock solution was aliquoted and stored at -80°C.

[0135] As described by Bremer et al. (2009) and Glebe and Gerlich (2004), HBV purification was performed via sucrose density gradient centrifugation. Briefly, HBV-infected patient plasma was loaded onto ultracentrifuge tubes containing a discontinuous sucrose gradient of densities of 60%, 45%, 35%, 25%, and 15%. The sucrose solution was prepared using TNE (20 mM Tris-HCl, 149 mM NaCl, 1 mM EDTA, pH 7.4). The plasma-loaded tubes were then centrifuged in an SW32 rotor (Beckman) at 112,000 g at 10°C for 15 hours. After centrifugation, the viral fraction (between 45% and 35%, determined by a real-time PCR detection kit (KHB, Shanghai, China)) was diluted in TNE and precipitated in an SW32 rotor at 112,000 g for 15 hours with 10–15% sucrose. The precipitate was resuspended in 2 ml of Null medium.

[0136] For CsCl density gradient purification, plasma was loaded onto a CsCl step gradient (five steps, 2 ml each) at densities ranging from 1.1 to 1.5 g / ml. After centrifugation at 174,000 g at 4°C for 35 hours in an SW41 rotor (Beckman), the gradient fractions were collected and the virus-containing fractions were detected by real-time PCR using a kit (KHB, Shanghai, China).

[0137] Hepatitis B virus infection

[0138] HBV infection occurs at a viral load of 200 genomic equivalents, or 1 x 10^6. 5 Cell seeding 2x10 7 A copy of the genome is equivalent to HBV. Fresh inoculum culture medium was prepared by initially dissolving PEG in null to a final PEG concentration of 4% and without adding DMSO. The inoculum volume was adjusted for plating (25 μl for 384-well plates, 100 μl for 96-well plates, and 500 μl for 24-well plates). After 20 hours, the inoculum was removed, and the cells were washed three times with PBS, followed by three more washes after 3 hours, and cultured under different conditions (Null / DMSO / 5C), with the medium changed every 3 days. Cells were cultured (without PEG, without DMSO) for a specified time before harvesting and further analysis. In some comparative experiments, inoculation was performed using MOI titration (10 / 50 / 100 / 200 / 400).

[0139] For reinfection, the supernatant of HBV-infected 5C-PHH was collected and concentrated 100 times by centrifugation at 3,000g for 1 hour at 4°C using a centrifugal filtration device (Amicon Ultra-15, regenerated cellulose 100,000, Merck Millipore Corp.), and then used to inoculate juvenile PHH according to the infection method described above.

[0140] Hepatitis B virus product testing

[0141] Following the manufacturer's instructions, HBV viral antigens HBsAg and HBeAg were detected using a commercial ELISA kit (Autobio, Zhengzhou, China) and 50 μl of cultured PHH supernatant.

[0142] To quantify HBV DNA, viral DNA was isolated using the DNeasy Blood & Tissue Kit (QIAGEN, catalog number: 69504). The isolated DNA was quantified by real-time PCR using a detection kit (KHB, Shanghai, China). The equivalent viral genome copies were calculated based on a standard curve generated using known copy numbers.

[0143] To quantify HBV-specific RNA, total RNA from HBV-infected cells was isolated using the RNeasy Plus Mini Kit (QIAGEN). Approximately 400 ng of total RNA was reverse transcribed into cDNA using the TransScript First-Strand cDNA Synthesis SuperMix (TransGenBiotech). Primers 5'-GAGTGTGGATTCGCACTCC-3' and 5'-GAGGCGAGGGAGTTCTTCT-3' were used for the HBV 3.5 kb transcript; primers 5'-TCACCAGCACCATGCAAC-3' and 5'-AAGCCACCCAAGGCACAG-3' were used for the total HBV-specific transcript. Amplification of the 123 bp fragment of the 3.5 kb transcript and the 92 bp product of the total HBV-specific transcript were performed by denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C denaturation and 60°C annealing / extension for 30 seconds at a rate of 3 seconds per cycle.

[0144] The cccDNA detection method used in this study was employed. Briefly, DNA was isolated using the DNeasy Blood & Tissue Kit (QIAGEN, catalog number: 69504). The extracted DNA was digested with plasmid-safe ATP-dependent DNase (Epicentre Technologies) at 37°C for 0.5 h, followed by inactivation at 70°C for 30 min. Then, 0.5 μl of 10 μl of the reaction mixture was added to 10 μl of a real-time PCR reaction. The viral genome equivalent copies were calculated based on a standard curve generated using known copy numbers. Power... Real-time PCR was performed using Green PCR Master Mix (Applied Biosystems) on a CFX384™ Real-Time PCR Detection System (Bio-Rad). For Southern Blot analysis of HBV cccDNA, HBV DNA was extracted using the core DNA extraction method and a modified Hirt DNA extraction method. For Southern Blot, the prepared DNA samples were separated on 0.8% agarose gels and then transferred to nylon membranes (Amersham). To confirm the identity of cccDNA, untreated and SpeI-digested 5C-PHH Hirt DNA preparations were separated on agarose gels. 1.7kbp, 2.1kbp, and 3.1kbp HBV DNA fragments containing labeled HBV probes (370bp-705bp HBV DNA fragments, Southern Blot probes) were also run on the same agarose gels as molecular markers in relaxed circular DNA (rcDNA), cccDNA, and single-stranded DNA (ssDNA) in Southern Blot analysis. Southern blotting was performed using the DIG HighPrime DNA Labeling and Detection Starter Kit II (Roche, 11 585 614 910). The primer sequences used to prepare the DNA marker (genotype D, kindly provided by Professor Li Wenhui) are as follows: HBV 3.2kb marker: HBV-D-EcorI-F 5'-GAATTCCACAACCTTTCACCAAA HBV-D-EcorI-R 5'-GAATTCCACTGCATGGCCTGAGGATGA HBV 2.1kb marker: HBV-D-EcorI-F 5'-GAATTCCACAACCTTTCACCAAA QYH-2.1kb-R 5'-CCCAGGTAGCTAGAGTCATTAGT HBV 1.7kb marker: HBV-D-EcorI-F 5'-GAATTCCACAACCTTTCACCAAA QYH-1.7kb-R 5'-AAGGTCGGTCGTTGACATTGCAGAGA

[0145] HCV infection and HCV product testing

[0146] The Huh7.5 cell line was kindly provided by Professor Charles Rice and routinely maintained in Dulbecco modified medium supplemented with 10% fetal bovine serum (Biological Industries), 25 mM HEPES (Gibco), and non-essential amino acids (Gibco). To generate HCV in cell culture, the plasmid encoding HCV, Jc1FLAG2(p7-nsGluc2A) (Jc1G in this text), was first linearized by XbaI digestion and used as a template for in vitro transcription using MEGAscript (Invitrogen). HCVcc were generated as previously described by electroporating the in vitro transcribed Jc1G RNA into Huh7.5 cells. Virus-containing supernatants were harvested daily for 3 days post-electroporation and up to 7 days post-electroporation. The supernatants were combined and used… The Ultra-15 (100K) centrifugal filter (Millipore) concentrates the virus by centrifuging at 5000g for 30 minutes at 4°C. This is achieved using... Repeated centrifugation with an Ultra-15 followed by replacement of the buffer with PBS. The concentrated virus was then titrated in a Huh 7.5 using a limiting dilution method.

[0147] To quantify intracellular HCV RNA, cells were lysed using Trizol reagent (Invitrogen). RNA was purified from the Trizol extract and then processed using PrimeScript. TM Reverse transcription was performed using the RT kit and TaKaRa's gDNA Eraser (Perfect RealTime) according to the manufacturer's instructions. Real-time PCR was then performed on the cDNA sample using primers targeting the listed specific genes. Premix Ex Taq TM Tli RNaseH Plus). GAPDH:(s)GGT ATC GTG GAA GGA CTCATG A,(as)ATG CCA GTG GCT TCC CGT TCA GC; HCV:(s)CCC TGT GAG GAA CTA CTG TCTTCA CGC,(as)GCT CAT GGT GCA CGG TCTA CGA GAC CT. Relative RNA levels were calculated using the 2-ΔΔCt method. GAPDH was used as a housekeeping gene for loading normalization.

[0148] To quantify luciferase activity, supernatant was removed from each well and mixed with an equal volume of 2× passive lysis buffer (Promega). Luciferase activity was then measured using Renegades luciferase substrate (Promega) according to the manufacturer's protocol.

[0149] Immunohistochemical staining and immunofluorescence staining

[0150] Cells were fixed in 4% paraformaldehyde (DingGuo, AR-0211) at room temperature for 20 minutes, then infiltrated and blocked with PBS containing 3% normal donkey serum (Jackson Immuno Research, 017-000-121) and 0.25% Triton X-100 (Sigma-Aldrich, T8787) at room temperature for 60 minutes. Cells were then incubated overnight with primary antibody at 4°C. Secondary antibody (Jackson Immuno Research) was applied and incubated at room temperature for 1 hour. Cell nuclei were stained with DAPI (Roche LifeScience, 10236276001). The primary antibodies used are listed in Table 3.

[0151] Table 3. Antibodies used for immunofluorescence staining.

[0152]

[0153] To detect the spread of HBV infection, immunohistochemical staining with HBcAg was performed. Briefly, cells were fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. After washing with PBS, cells were treated with a distilled aqueous solution of 50% ethanol, followed by a distilled aqueous solution of 70% ethanol at 4°C for at least 1 hour. After blocking (blocking solution: PBS with 10% FBS) for at least 2 hours, cells were incubated overnight at 4°C with anti-HBcAg (diluted 200-fold in blocking solution). After washing off the primary antibody, cells were post-primary treated with the primary antibody for 30 minutes at room temperature. Cells were then incubated with the secondary antibody for 30 minutes at room temperature. Fresh staining medium was prepared by diluting 50×DAB in DAB diluent, and then 150 μL of substrate solution was added to each well. After the DAB reaction, cells were counterstained with hematoxylin for 5–10 minutes.

[0154] qRT-PCR

[0155] Total RNA was isolated using the RNeasy Micro Kit (QIAGEN). RNA was converted to cDNA using the First-Strand Synthesis SuperMix (Invitrogen) for qRT-PCR. PCR was performed using Power... The qRT-PCR was performed using Green PCRMaster Mix (Applied Biosystems) on an MX3000P sequencing system (Stratagene). Data were analyzed using the Δ-ΔCt method. Primers used for qRT-PCR are listed in Table 4.

[0156] Table 4. Primers used for qRT-PCR.

[0157]

[0158]

[0159] RNA sequencing

[0160] Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN). RNA sequencing libraries were constructed using the Illumina mRNA-Seq Prep Kit (Illumina). Fragmented and randomly primed 150 bp paired-end libraries were sequenced using an Illumina HiSeq4000.

[0161] Software and algorithms used for transcriptome analysis included Cutadapt 1.16 (http: / / cutadapt.readthedocs.io / en / stable / guide.html), Salmon 0.8.2 (https: / / combinelab.github.io / salmon / ), and R 3.5.0 (https: / / www.r-project.org). Expression profiles of HepG2, F-PHH-3, and F-PHH-4 were downloaded as raw data. Expression profiles of PHH 0d, hiPSC-Hep rep1, hiPSC-Hep rep2, hiPSC-Hep rep3, and hiPSC-Hep rep4 (Gordillo et al., Development, 142:2094-2108(2015)) were downloaded as raw sequencing reads from the GEO databases (GSM2817112, GSM2753376, GSM2753377, GSM2753378, GSM2753379). To compare gene expression profiles between paired-end and single-end libraries, we used Reading 1 from our generated sequencing paired-end reads. Reading 1 and the downloaded single-end reads were resized to the same 40 bp length using Cut Adapt. Expression values ​​for each sample were quantified using Salmon. Unsupervised hierarchical clustering of the RNA-Seq data was generated using the hclust package in R.

[0162] Biochemical assay

[0163] Human albumin was measured using a human albumin ELISA quantitative kit (Bethyl Laboratories). Urea concentration was measured using QuantiChrom. TMUrea assay kit (BioAssay Systems). Measure according to the manufacturer's instructions. The Invitrogen MTT Cell Proliferation Assay Kit performs MTT assays at specified time points.

[0164] Statistical analysis

[0165] For statistical analysis, a two-tailed unpaired t-test was used. Results are expressed as mean ± SEM, where n represents the number of replicates from the same PHH batch. p-values ​​are as follows: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0166] Results and discussion

[0167] To maintain hepatocyte-hepatitis H (PHH) long-term in vitro, a novel chemical strategy using combinations of small molecules was developed. Small molecules, due to their ability to achieve flexible spatiotemporal tuning of specific cellular targets, possess the advantage of orchestrating innate signaling in cultured cells. To identify small molecules that promote PHH maintenance, RNA sequencing (RNA-Seq) was first used to compare the global gene expression profiles of freshly isolated primary human hepatocytes (F-PHH) with those of hepatocytes cultured in vitro for only 24 hours. Consistent with previous studies, key hepatocyte functional genes were significantly downregulated after 24 hours of culture. Figure 1 Notably, the expression of key components of the TGF-β signaling pathway was upregulated in hepatocytes cultured for 24 hours. Figure 2A ).

[0168] Previous studies have shown that TGF-β signaling is a major inducer of epithelial-mesenchymal transition (EMT) in hepatocytes. Therefore, the TGF-β inhibitor SB431542 (SB43) was used to block TGF-β signaling-induced EMT, and cultured hepatocytes treated with SB43 maintained their epithelial morphology and expressed E-cadherin even after 2 weeks (data not shown). However, blocking TGF-β signaling failed to inhibit the expression of EMT-related transcription factors Snail Family Transcriptional Repressor 2 (SNAI2); Twist-associated protein 1 (TWIST1); and the expression of rescue liver function genes (ALB; CPS1 (carbamoyl phosphate synthase-1); CYP3A4 and CYP1A2). Figure 2B This indicates that blocking TGF-β signaling partially inhibits EMT, but has limited effect on maintaining the functional state of PHH.

[0169] To identify other signaling pathways crucial for maintaining functional hepatitis B (PHH), a small molecule library was screened by adding individual small molecules in combination with SB43 to cultured hepatocytes. The small molecules targeted included fibrinogen (FSK), an activator of adenylate cyclase (AC), gene expression regulators of sustained liver function (ALB; CSP1; CYP3A4; OATP1B1), and downregulatory EMT marker gene expression α-smooth muscle actin (ASMA). Figure 3A and 3B This combination of FSK and SB43 maintained liver morphology and sustained PHH survival. Under these two chemical conditions containing both FSK and SB43 (referred to here as "2C"), we observed that hepatocytes exhibited typical liver polarized polygonal morphology and rare cell death after two weeks of culture. In contrast, without FSK and SB43, hepatocytes became morphologically depolarized within two weeks, and cell viability in culture was very low.

[0170] PHH (2C-PHH) maintained under 2C conditions for an extended period exhibits the functional properties of its freshly isolated counterpart.

[0171] A study was conducted to test whether 2C conditions could sustain PHH function long-term. First, the expression of a set of key liver function markers was assessed, including serum proteins synthesized and secreted by the liver (ALB and ApoC1), rate-limiting enzymes of nitrogen metabolism (NAGS, CPS1, and OTC), key heterologous substance-metabolizing cytochrome P450 (CYP) enzymes (CYP3A4, CYP2D6, and CYP2C9), and transport proteins (NTCP, MRP2, and BSEP). RT-qPCR analysis showed that the expression of these genes was comparable between 2C-PHH and F-PHH. Figure 4B Further analysis was conducted on a broad spectrum of other liver function genes, such as those involved in amino acid and fatty acid metabolism and coagulation. The results showed that the gene expression profile of 2C-PHH was similar to that of F-PHH. To further confirm that 2C is functionally independent of the basal culture medium type, 2C was tested using both WME and HCM. RT-qPCR analysis showed that after 4 weeks of culture, 2C-PHH treated with 2C in both HCM and WME expressed liver genes at levels comparable to those of F-PHH. Figure 4C (Figure at the bottom). This result indicates that the role of 2C is independent of culture medium type, and the following experiments are based on WME. Importantly, the combination of FSK and SB43 showed a synergistic effect on hepatocyte maintenance, possibly by activating cAMP (measured as upregulation of ALB, N-acetylglutamate synthase (NAGS), apolipoprotein CI (APOC1), CYP2D6, and NTCP expression in FSK+SB43 treatment compared to FSK or SB43 treatment alone; and downregulation of SNAI2 and TWIST1). Figure 4A -D).

[0172] In summary, these data indicate that 2C conditions effectively maintain the expression of key liver function genes in long-term cultured PHH.

[0173] Next, albumin (ALB) secretion, representing protein synthesis and secretion capacity, and urea synthesis, representing nitrogen metabolism capacity in human hepatocytes, were quantified. In hepatocytes without 2C, ALB secretion decreased sharply within one week but remained well for approximately four weeks in 2C-PHH. Similar results were obtained for urea synthesis. These results were reproducible on different batches of PHH. Figure 4E ).

[0174] The in vivo implantation capacity of 2C-PHHs was tested. After 4 weeks, 2C-PHHs robustly refilled recipient mouse livers at levels comparable to F-PHHs and secreted human serum albumin (HSA). In contrast, hepatocytes cultured without 2Cs struggled to refill mouse livers, as evidenced by the fact that HSA was undetectable 2 weeks after transplantation and almost undetectable at 4 weeks compared to 2C-PHHs. Figure 4J Overall, these data suggest that 2C can support the maintenance of PHH function in vitro for at least 4 weeks.

[0175] Maintenance of drug metabolism capacity in hepatocytes cultured under 2C conditions

[0176] To assess the drug metabolism capacity of 2C-PHH, the expression of key CYP enzymes was quantified by RT-qPCR. After one month of culture, the expression levels of CYP3A4, CYP1A2, CYP2C9, CYP2D6, CYP2B6, and CYP2C19 in 2C-PHH were comparable to those in F-PHH. Figure 4K To further evaluate drug metabolic activity, liquid chromatography-mass spectrometry (LC / MS) was used for determination. Data showed that after one month of culture, the metabolic activities of CYP3A4, CYP2D6, CYP2C9, and CYP1A2, responsible for over 80% of drug oxidation in humans, in 2C-PHH were comparable to those in F-PHH, while hepatocytes without 2C culture lost these metabolic activities. Figure 4F ).

[0177] Further experiments were conducted to test whether 2C-PHH could be used to predict drug-drug interactions (DDIs). Potent CYP enzyme inhibitors and inducers commonly used in DDI testing (Baranczewski et al., PR, 58:453-472, (2006)) were applied, and the results showed that 2C-PHH responded effectively to these inhibitors and inducers. Figure 4G , 4H And 4I). Specific inhibitors of CYP enzymes significantly inhibited the drug metabolism activity of 2C-PHH (and 4I). Figure 4I Furthermore, after one month of culture, the gene expression levels and metabolic activities of the three major CYP enzymes CYP3A4, CYP1A2, and CYP2B6 in 2C-PHH responded well to specific inducers. Figure 4G and 4H In summary, these data suggest that 2C can support the maintenance of PHH's in vitro drug metabolic activity.

[0178] Recovered hepatocytes maintained in cryopreservation using 2C with additional small molecules

[0179] Given that 2C enables PHH to maintain function robustly for extended periods, we then tested whether 2C could support the maintenance of cryopreserved primary human hepatocytes (frozen PHH) (which is a major resource of human hepatocytes in application). However, frozen PHH is more difficult to maintain stably in vitro due to poor viability and function caused by the cryopreservation process (Donato et al., Current Drug Metabolis, 9:1-11 (2008b); Terry et al., Mol. Cell Transplantation, 16:639-647 (2017)). Under 2C conditions, we observed improved survival of cultured frozen PHH. However, they lost their polygonal morphology after 4 weeks of culture (data not shown), indicating that EMT occurred in 2C-treated frozen PHH.

[0180] Based on this combination of SB43 and FSK, further chemical screening was conducted, and the results showed that DAPT (Notch inhibitor), IWP 2 (Wnt inhibitor), and LDN193189 (BMP inhibitor) further blocked the expression of EMT marker genes (collagen type I α1 (COL1A1); thymocyte antigen 1 (Thy1); vimentin (VIM)). Figure 5A -C). It is noteworthy that small molecules or cytokines targeting the same signaling pathway lead to similar repression of EMT genes ( Figure 5D This minimal condition, consisting of five chemicals (5C)FSK, SB43, DAPT, IWP 2, and LDN193189, effectively blocked the expression of the EMT gene in cultured cryopreserved primary human hepatocytes (frozen PHH). Figure 6A and 5E(Expression of COL1A1, a typical biomarker of mesenchyme, was effectively downregulated after 5C treatment), and this supported the maintenance of the typical polygonal shape of F-PHH and frozen PHH for 4 weeks (data not shown). Furthermore, 5C-cultured PHH (5C-PHH) expressed surrogate liver function markers (ALB, AAT (α1-antitrypsin), CPS1, CYP3A4; CYP1A2), and exhibited polarization after 4 weeks of culture, measured as CDFDA deposition at the cell-cell boundary (data not shown). Albumin secretion and urea synthesis in 5C-PHH were also maintained for at least 3 weeks and were reproducible on 9 batches of PHH. Figure 6B-6E Interestingly, the PHH batch with high survivability can maintain its functionality for 2 months under 5C conditions.

[0181] Next, within one month of isolation, a time-series analysis of the global gene expression profile of 5C-PHH was performed using RNA-Seq. A similar analysis was also performed for comparison using the sandwich culture method (i.e., culturing hepatocytes between two layers of gel collagen) (a commonly used hepatocyte culture condition) (Dunn et al., Biotechnol. Prog., 7:237-245 (1991)). Hierarchical clustering showed that, at extended culture times of 12 hours, only 5C-PHH clustered with F-PHH, while the transcriptome of PHH in sandwich culture was similar to that of the Null and DMSO groups. Notably, sandwich cultures did not cluster with 5C. The ability of the 5C condition to support global hepatic gene expression was reproducible in three other independent batches of PHH. Figure 7 Furthermore, qRT-PCR analysis further confirmed that, compared with the sandwich method, the 5C condition was more effective in maintaining the expression of alternative hepatocyte functional markers and inhibiting the expression of mesenchymal marker genes. Figure 8A Furthermore, 5C effectively maintains the expression of transcription factors that play a key role in hepatocyte function. Figure 8B In summary, these results indicate that 5C conditions can maintain the global gene expression pattern of human hepatocytes in vitro for an extended period.

[0182] Hepatocytes are known to play a crucial role in the metabolism and detoxification of exogenous drugs. However, the metabolic activity of hepatocyte CYPs is rapidly lost during culture. Figure 1 In the publicly available 5C system, PHH cultured for 4 weeks was found to express core CYP enzymes at levels comparable to F-PHH. Furthermore, the expression levels of major phase II drug-metabolizing enzymes and phase III drug-metabolizing transporters in 5C-PHH were similar to those in F-PHH. Figure 8D and 8E These data indicate that 5C effectively preserves the expression of drug metabolism genes.

[0183] Then, key CYP enzymes responsible for approximately 80-90% of clinical drug metabolism in the human body were characterized, including CYP3A4, CYP1A2, CYP2C9, CYP2D6, and CYP2B6. Liquid chromatography-mass spectrometry analysis showed that, after 2 weeks of culture, the metabolic activities of these major enzymes in 5C-PHH were comparable to those in F-PHH. Figure 8F In contrast, the metabolic activity of PHH cultured in the control group was almost undetectable. Figure 8F These results were reproducible on three different batches of PHH. Overall, these results indicate that 5C maintains the drug metabolic activity of cultured human hepatocytes in vitro.

[0184] The robust functional maintenance of human hepatocytes under 5C culture conditions provides a platform for in vitro HBV infection modeling. qRT-PCR analysis confirmed that the NTCP expression levels of F-PHH and PHH cultured for 4 weeks under 5C conditions were comparable. Figure 9A Next, successful HBV infection was achieved by inoculating PHH with plasma from HBV-infected patients, and HBV products (secreted HBsAg, HBeAg, and DNA particles) were detected in the supernatant of 5C-PHH cultures. Figure 9B and 9D Intracellular HBV-3.5kb and total HBV RNA transcript levels also increased over time. Figure 9C In 5C-PHH, high levels of HBV product production persisted for at least one month. Importantly, the formation of HBV cccDNA was detectable in 5C-PHH by Southern Blot. Figure 9H qPCR analysis showed that cccDNA remained detectable after 4 weeks of culture at 5C. Figure 9E Immunostaining with HBV core antigen (HBcAg) demonstrated the effectiveness of 5C-PHH in HBV infection (data not shown). Furthermore, 5C-PHH remained susceptible to HBV infection even when the MOI decreased to 10. Figure 10A and 10B 5C-supported HBV infection was well reproduced in additional batches (>5 batches) of PHH (including frozen PHH). Figure 11A -E). Importantly, 5C stably supports HBV infection from a variety of sources, including various HBV genotypes and those from the in vitro production cell line HepAD38 ( Figure 11A -D) infection.

[0185] Viral particles were collected from the supernatant of HBV-infected 5C-PHH and inoculated with them into immature PHH. HBeAg and HBV DNA were detected in the PHH inoculated with the supernatant of HBV-infected 5C-PHH. Figure 9F The presence of 5C-PHH (-G) indicates that 5C-PHH can support the entire HBV life cycle and generate HBV infectious particles. Furthermore, the proportion of HBV-infected 5C-PHH increases with increasing culture time (data not shown). Figure 11E This indicates that HBV may be transmitted through intercellular transmission and / or de novo infection of HBV particles.

[0186] The 5C-PHH infection model was found to be sensitive to two classes of clinical anti-HBV drugs: interferon-α (IFN-α) and nucleotide reverse transcriptase inhibitors, such as entecavir (ETV) and lamivudine (LAM). Treatment of HBV-infected 5C-PHH with all three drugs (ETV, LAM, and IFN-α) resulted in a reduction in HBV production. Figure 12A Similar results were obtained from cryopreserved PHH cultured in 5C, where IFN-α led to a significant reduction in cccDNA. Furthermore, although interferon-stimulated genes (ISGs) were significantly upregulated in long-term cultured 5C-PHH treated with IFN-α, they responded weakly to HBV infection, consistent with previous reports of HBV evading hepatocyte innate immunity. Figure 13A ) (Cheng et al., Hepatol., 66:1779-1793 (2017)).

[0187] Further comparison of culture conditions with other reported culture conditions for long-term HBV infection in PHH (Lucifira et al., Science, 343:1221-1228 (2014), Winner et al., Nat. Comm., 8:1256 (2017), Xia et al., J. Hepatol., 656:494-5603 (2017)) revealed that 5C showed a significant advantage, especially in maintaining hepatocyte function and stably supporting HBV infection in the long term (data not shown). Figure 13B , 13C And 13D). Importantly, the 5C condition can be scaled down to micropore form, suitable for high-throughput applications (and 13D). Figure 14A These results further establish 5C as a simple, efficient, and stable culture condition for long-term maintenance of PHH in vitro, which is of great value for the study of HBV infection.

[0188] Besides HBV, 5C-PHH can also be infected with other hepatitis viruses, such as hepatitis C virus (HCV). 5C-PHH was exposed to HCV Jc1G infectious viral particles expressing a secretory Gaussian luciferase (Gluc) reporter gene. Viral replication was detected in 5C-PHH and was effectively blocked by DCV (a direct-acting antiviral drug used to treat hepatitis C). Figure 15AUnlike HBV, HCV induces strong ISG expression in 5C-PHH. Figure 15C In summary, these results indicate that 5C-PHH can serve as an effective model for modeling hepatotropic infections and investigating antiviral strategies.

[0189] In summary, a chemical approach has been developed to maintain the function of primary human hepatocytes long-term. Using this platform, persistent HBV infection was demonstrated, which extended throughout the viral life cycle and released viral progeny in vitro at a PHH (Probiotic-Hydrogen-Hepatic Acid) environment. Notably, the formation of HBV cccDNA was captured in the PHH, a key factor in HBV re-entry into the viral life cycle and induction of HBV relapse in human liver. This platform is simple and easily applicable to high-throughput screening for designing novel antiviral strategies, particularly for cccDNA-targeting compounds, which may prove crucial for finding a cure for chronic hepatitis. Methodologically, the small molecules possess highly tunable functions, precisely balancing cellular signaling and modulating molecular networks to stabilize cellular identity and function in mature cells; this strategy can be extended to the maintenance of other functional cell types.

[0190] Global gene expression in hepatocytes cultured under 2C and 5C conditions

[0191] To monitor dynamic gene expression changes in hepatocytes (PHHs) throughout culture, hepatocytes from two different batches (B1 and B2) cultured with and without 2C and 5C were harvested at days 1.5, 3, 5, 8, 15, and 27 for gene expression profiling analysis via RNA-Seq. F-PHH and frozen PHH were used as positive controls. Hierarchical clustering showed that 2C-PHH and 5C-PHH had global gene expression profiles similar to their freshly isolated counterparts (data not shown). Consistent with functional maintenance, the results showed that 2C and 5C conditions maintained the expression of key transcription factors (TFs) in hepatocytes at levels comparable to those of F-PHH (data not shown). These data suggest that 2C / 5C supports the long-term maintenance of a global gene expression profile in cultured PHHs similar to that of F-PHH.

[0192] Both cAMP-PKA and cAMP-EPAC are essential for maintaining PHH.

[0193] To investigate the potential mechanisms under our chemical conditions, the core small molecule combination maintaining human hepatocytes, FSK and SB43, was replaced by analogs that block or stimulate the same signaling pathways. Simultaneously, gene expression libraries of cultured hepatocytes were analyzed by RNA-Seq. Hierarchical cluster analysis revealed that FSK plays a crucial role in maintaining PHH (data not shown). NKH477 (a cAMP agonist) and db-cAMP (a cAMP analog) replaced FSK to maintain global gene expression in PHH. In contrast, hepatocytes treated with 1,9-ddeoxycholangin (1,9-dFSK) (an inactive analog of FSK) induced expression profiles similar to the control group without FSK. The effects of db-cAMP on hepatic marker gene expression, ALB secretion, and urea synthesis were investigated (data not shown). Data indicate that FSK is an essential small molecule for PHH maintenance, functioning by activating AC (data not shown).

[0194] Further investigation was conducted into the downstream targets of cAMP. Results showed that inhibition of either PKA or EPAC alone promoted the expression of mesenchymal markers, indicating EMT occurring during hepatocyte culture (data not shown). Furthermore, activation of either PKA or EPAC alone could not fully compensate for the withdrawal of FSK from cell line 2C (data not shown). These data suggest that cAMP may be a central signaling hub for the PKA, EPAC, and downstream cAMP pathways, acting synergistically but non-redundantly in PHH maintenance.

[0195] Chemical mixtures promote the functional maturation of hESC-derived hepatocytes

[0196] Given that 2C can maintain the maturation function of human hepatocytes, a study was conducted to test whether the published chemical culture conditions could promote the maturation of human embryonic stem cell-derived hepatocytes (hESC-Hep). hESC-Hep was generated using a modified version of an established protocol (Zhao et al., Cell Res., 23:157-161 (2013)). Immunofluorescence analysis showed that hESC-Hep expressed hepatocyte-specific TFs FOXA2, HNFA4, HNF6, PROX1, and CEBPA (data not shown). Albumin was detected in hESC-Hep. hESC-Hep was immunopositive for AFP and ALB (data not shown) and CYP3A4, but not for CYP1A2, CYP2C9, CYP2A6, or CYP2E1 (data not shown). These data suggest a fetal-like functional profile of hESC-Hep.

[0197] In contrast, after hESC-Hep was transferred to 2C conditions, it acquired a polygonal morphology with a low nucleus-to-cytoplasm ratio (data not shown). Furthermore, liver function marker genes (HNFA4, HNF6, PROX1, CEBPA, CYP2C9, CYP2A6, CYP1A2, etc.) were gradually upregulated from day 7 in 2C culture (data not shown). Figure 16 Notably, intercellular bile canaliculi formed after 2 weeks of culture (data not shown) and AFP expression decreased (data not shown). Importantly, 2C-cultured hESC-Hep expressed a range of CYP enzymes, including CYP3A4, CYP1A2, CYP2C9, CYP2C19, CYP2A6, and CYP2E1 (data not shown). Two key nuclear receptors related to drug metabolism, PXR and CAR, were significantly upregulated under 2C conditions (data not shown). Figure 16 Under 2C conditions, expression of HNF4A, the major regulator of hepatocyte-specific genes in adults, was observed, along with increased expression of maturation factors including CEBPA, PROX1, and ATF5. Otherwise, hepatocyte-specific genes gradually decreased without 2C treatment. Figure 16 In summary, these data suggest that publicly available adult hepatocyte chemistry conditions promote functional maturation of hPSC-Hep, closely resembling the functional maturation state of adult hepatocytes.

Claims

1. A method for long-term in vitro maintenance of functional hepatocyte function, comprising culturing hepatocytes in a culture medium supplemented with a hepatocyte cell culture composition, wherein the composition contains effective amounts of an adenylate cyclase activator and a TGFβ inhibitor to maintain functional hepatocyte function in vitro for a long period of time, as measured below: compared with hepatocytes cultured in an unsupplemented culture composition without the adenylate cyclase activator and the TGFβ inhibitor, the expression of at least one hepatocyte marker is increased after in vitro culture of hepatocytes in supplemented cell culture composition for at least two weeks; in, The at least one hepatocyte marker is selected from albumin ALB, N-acetylglutamate synthase NAGS, apolipoprotein CI, CYP2D6, or NTCP.

2. The method of claim 1, wherein the composition further comprises at least one Notch inhibitor, a Wnt inhibitor, and a BMP inhibitor.

3. The method of claim 2, wherein the composition comprises a combination of at least one adenylate cyclase activator, a TGFβ inhibitor, a Notch inhibitor, a Wnt inhibitor and a BMP inhibitor.

4. The method according to any one of claims 1-3, wherein the adenylate cyclase activator is trichosine.

5. The method according to any one of claims 1-3, wherein the TGFβ inhibitor is SB431542.

6. The method according to any one of claims 2-3, wherein the Notch inhibitor is DAPT.

7. The method according to any one of claims 2-3, wherein the Wnt inhibitor is IWP 2.

8. The method according to any one of claims 2-3, wherein the BMP inhibitor is LDN193189.

9. The method according to any one of claims 1-3, wherein the hepatocytes express at least six hepatocyte functional proteins after being cultured for at least 60 days, at levels identical to those of freshly isolated primary hepatocytes (F-PHH) from the same organism.

10. The method according to any one of claims 1-3, wherein the hepatocytes express at least six hepatocyte functional proteins at levels identical to those of F-PHH from the same organism after being cultured for at least 90 days.

11. The method according to any one of claims 1-3, wherein the hepatocytes, after being cultured in vitro for at least two weeks, have at least one characteristic selected from the group consisting of: polygonal hepatocyte morphology, established hepatocyte function, and expression of at least one known hepatocyte marker.

12. The method according to claim 11, wherein, The established hepatocyte functions include albumin secretion and urea synthesis.

13. The method according to any one of claims 1-3, wherein the hepatocytes, after being cultured for 3 to 8 weeks, have at least one characteristic selected from: polygonal hepatocyte morphology, established hepatocyte function, and expression of at least one known hepatocyte marker.

14. The method according to any one of claims 1-3, wherein the hepatocytes express at least one of the listed hepatocyte markers at a comparable level or more than that measured as statistically insignificantly different from the level expressed by freshly isolated hepatocytes from the same organism.

15. The method according to any one of claims 1-3, wherein the hepatocytes, after being cultured for 90 days, express at least one of the following hepatocyte markers at a level comparable to that of freshly isolated hepatocytes obtained from the same organism: APOA2, APOB, F2, F10, CYP3A4, CYP1A2, CYP2C9, and UTG2B7.

16. The method according to any one of claims 1-3, wherein the hepatocytes, after 2 weeks of culture, contain metabolic activities of CYP3A4, CYP1A2, CYP2C9, CYP2D6 and CYP2B6 comparable to those of F-PHH.

17. A method for preparing an in vitro liver parasitic infection model, comprising: The duration of liver cells cultured according to any one of claims 1-8 that remain effective against the parasitic organism is determined by inoculating them with the liver parasite, and the parasite-infected cells are cultured in vitro in a liver basal medium supplemented with HC.

18. The method of claim 17, wherein the parasitic organism is a hepatitis virus, the method comprising inoculating hepatocytes cultured according to any one of claims 1-8 with the hepatitis virus for a duration effective in infecting the virus, and culturing the virus-infected cells in vitro in a liver basal medium supplemented with HC.

19. The method of claim 18, wherein the hepatitis virus is hepatitis B, hepatitis D, or hepatitis C.

20. The method of claim 19, wherein the HBV covalently closed circular DNA (cccDNA) is contained when cultured in vitro for 4 weeks.

21. The method of claim 17, wherein the parasitic organism is a Plasmodium species selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.

22. A culture medium comprising a basal culture medium and a supplemented hepatocyte cell culture composition, wherein, The supplementary hepatocyte culture composition consists of an effective amount of adenylate cyclase activator and a TGFβ inhibitor; wherein the concentration of the adenylate cyclase activator is 5-50 μM and the concentration of the TGFβ inhibitor is 2-15 μM. To maintain the function of functional hepatocytes in vitro for a long period of time, as measured below: compared with hepatocytes cultured in an unsupplemented culture composition without adenylate cyclase activator and TGFβ inhibitor, the expression of at least one hepatocyte marker was increased after in vitro culture in a supplemented cell culture composition for at least two weeks. The at least one hepatocyte marker is selected from albumin ALB, N-acetylglutamate synthase NAGS, apolipoprotein CI, CYP2D6, or NTCP.

23. The culture medium according to claim 22, wherein the adenylate cyclase activator is trichomoniasis.

24. The culture medium according to claim 22, wherein the TGFβ inhibitor is SB431542.

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