Search Results (35)

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a widespread condition with a complex pathogenesis. Cell-based models are important tools for studying the mechanisms underlying its development and progression. The aim of this review is to analyze the HepaRG, Huh-7, immortalized human hepatocyte (IHH), and primary human hepatocyte (PHH) cell lines for modeling and studying MASLD. HepaRG represents the most metabolically competent immortalized hepatocyte model with preserved biotransformation activity and a physiological bioenergetic response to lipid loading, making it valuable for pharmacological and toxicological studies. Huh-7 is distinguished by its accessibility and suitability for studying steatosis, lipotoxicity, insulin resistance, and paracrine mechanisms of fibrogenesis; however, its use is limited by its tumor origin, impaired carbohydrate metabolism, and low activity of xenobiotic-metabolizing enzymes. The IHH model occupies an intermediate position because of its non-tumor origin and is of interest for studies of senescence, epigenetic regulation, and signaling pathways involved in steatosis, although interpretation of results requires consideration of immortalization-related effects and specific metabolic limitations. PHH remains the most physiologically relevant platform for MASLD modeling, particularly in three-dimensional (3D) and microphysiological formats; however, its use is limited by high cost, interindividual variability, and the limited duration of the differentiated phenotype. Increasing model complexity—from two-dimensional (2D) monocultures to co-cultures, spheroids, and organ-on-chip systems—enhances physiological relevance and enables reproduction not only of steatosis but also of the inflammatory and fibrogenic components of MASLD progression, yet it reduces reproducibility and complicates standardization. Overall, none of the existing models is universal, and the optimal strategy is to select models according to the specific research question. A key direction for future research is the standardization of steatosis induction protocols and the unification of criteria for evaluating results. Full article

Background and Aims: Understanding regulatory interactions between hepatitis B virus (HBV) and host factors is essential for the development of next generation host-directed antiviral therapies and the achievement of a functional HBV cure. Here, we investigated HBV-induced alterations in host gene expression in primary human hepatocytes (PHH) to identify host factors exploited by the virus for replication and persistence. Whole-transcriptome sequencing (WTS) of HBV-infected PHH identified host pathways with potential roles in the HBV life cycle. RNA interference-based functional screening of dysregulated candidate genes identified cyclin L1 (CCNL1) as a key host factor. RNAi-mediated knockdown of CCNL1 reduced HBV gene expression, including hepatitis B surface antigen (HBsAg). Mechanistically, CCNL1 regulates phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII) at serine 2 (S2), consistent with a role in transcriptional regulation. CCNL1 knockdown further reduced the binding of total and phospho- (Ser2/Ser5) RNAPII, pan-acetylated histone H3 (H3ac), and H3K27ac to HBV covalently closed circular DNA (cccDNA), indicating impaired cccDNA-dependent transcription. In addition, CCNL1 expression was elevated in chronic hepatitis B patients compared with those with resolved infection. Collectively, these data demonstrate that CCNL1 promotes HBV transcription and replication through modulation of RNAPII phosphorylation and chromatin-associated transcriptional activity, identifying CCNL1 as a potential host susceptibility factor for HBV. Importance: Hepatitis B virus infection remains a major threat to human health in areas with high prevalence. There is need to fully understand the complex interactions between the virus and human host factors/processes to support ongoing efforts to develop anti-HBV therapies that can be used with existing therapies to achieve a better cure. HBV relies on host cellular factors and biological processes to establish and maintain efficient infection, making host–virus interactions attractive targets for therapeutic intervention. Thus, identifying host factors that support and/or restrict HBV infection is essential for understanding the molecular basis of chronic HBV infection and for developing host-targeting anti-HBV drugs. This study identifies cyclin L1 (CCNL1) as a host susceptibility factor that promotes HBV transcription and replication through regulation of RNA polymerase II activity and or post-transcriptional mechanisms. Full article

(1) Background: Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of viruses that interact with hepatocytes. HBV infection is a major global health problem. Most adults clear the infection quickly after being infected with HBV, while a few people develop chronic HBV infection. It is well-known that the early innate immune response of host cells plays an important role in the fight against virus infection. However, the interactions between HBV and the intrinsic innate immune system of hepatocytes are still not fully understood. The aim of this study was to confirm the interaction between HBV and hepatocytes, and to identify the interferon-stimulated genes (ISGs) regulated by HBx and their expression in association with HBV-associated HCC (HBV-HCC), so that we can refine our understanding of the interaction between HBV and ISGs and its potential influence on HBV-HCC. (2) Methods: We analyzed data concerning the stimulation of IFN-dependent genes in primary human hepatocytes (PHHs) transfected with pathogen DNA mimetics or infected with HBV in the GSE69590 database. Bioinformatic methods, such as GSEA, GO, and KEGG, were used to analyze the differentially expressed innate immunity genes and their related pathways to identify candidate intrinsic innate immune factors. qPCR on HepG2 and Huh7 cells, which highly express HBx, was used to detect relevant intrinsic innate immune factors. qPCR, RNAi, and Elisa methods were used to identify intrinsic innate immune factors in HBV-integrated HepG2.2.15 cells, and bioinformatics analysis was conducted on the HBV-infected tissues and cells in the GEO database. (3) Results: Inhibition of the JAK-STAT pathway enhanced HBV replication in HepG2 cells transfected with HBV plasmid and HepG2-NTCP cells infected with HBV. GSEA analysis of the GSE69590 data revealed significant changes in intrinsic innate immune pathways during HBV infection with PHH for 40 h. A total of 84 differentially expressed, candidate innate immunity genes were identified in GSE69590. Validation showed that TRIM22 and TRIM56 were down-regulated when HBx was expressed. Consistently, TRIM22 and TRIM56 were up-regulated following inhibition of HBx by transfection of HBx siRNA into HepG2.2.15 cells, and HBV pgRNA was up-regulated following down-regulated expression of TRIM22 and TRIM56 in HEK293 cells. Receiver operating characteristics (ROC) and overall survival (OS) analysis of 204 HBV-HCC patients showed that expression of TRIM22 was closely associated with HBV-HCC, and high expression of TRIM22 was associated with longer survival. (4) Conclusions: Innate immunity genes TRIM22 and TRIM56 are regulated by HBx, and higher expression of TRIM22 is closely related to longer survival of HBV-HCC patients. Full article

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a sexually dimorphic condition, with higher prevalence in men than in women. Sex differences in hepatic lipid metabolism and the modulatory role of sex hormones have been described but are still insufficiently understood. The aim of this study was to introduce the variables sex and sex hormones into a human in vitro model of hepatic steatosis. Methods: Primary human hepatocytes (PHHs) were isolated from male and female donors, treated with free fatty acids (FFA) to induce steatosis, and further exposed to physiological concentrations of estrogen, progesterone, or testosterone. Intracellular triacylglyceride (TAG) content, lipid droplet (LD) formation, FFA uptake, and very-low-density lipoprotein (VLDL) excretion were assessed. In parallel, the expression of lipid metabolism-related genes was quantified by qPCR. Results: FFA treatment induced comparable uptake and intracellular TAG storage in both sexes. However, female PHHs secreted approximately twice as many VLDL particles as male cells. Steatosis significantly increased expression of LDLR, CPT2, and PLA1A only in male PHHs. Sex hormones exerted distinct, sex-specific effects: estrogen reduced TAG accumulation in female PHHs; whereas testosterone reduced TAG in male but increased it in female PHHs after prolonged treatment. LD characterization confirmed sex- and hormone-dependent differences in lipid storage patterns. In male PHHs, progesterone promoted lipid storage and increased apoB-100 secretion, accompanied by reduced LDLR and APOA5 expression, and testosterone increased the FFA-mediated CPT2 even further. Conclusions: Sex and sex hormones distinctly shape hepatocellular lipid handling under steatotic conditions. While female PHHs demonstrated greater lipid excretion capacity, male PHHs exhibited stronger transcriptional responses. Sex-specific responses to estrogen and testosterone resembled clinical observations on sex hormone effects. These findings highlight the need to account for sex-specific differences in MASLD pathophysiology and therapeutic strategies. Full article

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a growing health concern worldwide. Human cell-based in vitro culture models that retain disease-relevant phenotypic pathways and responses to assess the efficacy and liability of new therapeutics are needed. Obeticholic Acid (OCA), a Farnesoid X Receptor agonist, has been identified for MAFLD treatment, and clinically shown to have anti-inflammatory and anti-fibrotic effects. In this study, healthy and disease-origin primary human hepatocytes (PHHs) were cultured in TruVivo^®, an all-human hepatic system for 14 days and treated with OCA to determine its’ effects on lipogenic, inflammatory, and fibrogenic pathways. Decreases in lipogenesis and triglyceride levels were measured in OCA treated healthy and diseased PHHs. Significant decreases in CYP3A4 activity and gene expression were quantified. Macrophage marker expression, pro-inflammatory cytokines and fibrotic markers were lowered in OCA treated diseased PHHs. CYP7A1 gene expression decreased, while BSEP gene expression increased in OCA treated healthy and diseased PHHs. Overall, OCA treatment reduced lipogenic, inflammatory, and fibrogenic markers in diseased PHHs. Differences in the potency and efficacy of OCA against different disease-relevant pathways were observed in healthy and diseased PHHs indicating divergence of key regulatory mechanisms between healthy versus diseased phenotypes. Full article

Human-induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) have been shown to be useful for the development of cell-based regenerative strategies and for modelling drug discovery. However, stem cell-derived HLCs are not identical in nature to primary human hepatocytes (PHHs), which could affect the cell phenotype and, potentially, model reliability. Therefore, we employed the in-depth gene expression profiling of HLCs and other important and relevant cell types, which led to the identification of clear similarities and differences between them at the transcriptional level. Through gene set enrichment analysis, we identified that genes that are critical for immune signalling pathways become downregulated upon HLC differentiation. Our analysis also found that TAV.HLCs exhibit a mild gene signature characteristic of acute lymphoblastic leukaemia, but not other selected cancers. Importantly, HLCs present significant similarity to PHHs, making them genuinely valuable for modelling human liver biology in vitro and for the development of prototype cell-based therapies for pre-clinical testing. Full article

Primary human hepatocytes (PHHs) are widely used as in vitro models for liver function and drug metabolism studies, yet their metabolic stability post-thawing remains an open question. To better characterize early metabolic changes, we conducted a time-course experiment using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze metabolic shifts in PHHs cultured in suspension. Unexposed and exposed (acetaminophen-treated) samples were evaluated, and TITAN analysis was applied to determine the time point of maximal metabolic change at both individual metabolite and global metabolic profile levels. Our results indicate that the majority of metabolic shifts occur within the first five hours post-thawing. In the early culture time points, substantial metabolic overlap was observed between unexposed and exposed cells, suggesting a conserved biological response likely related to cellular recovery. However, at later time points, metabolite profiles diverged, with acetaminophen treatment-specific metabolic changes emerging, potentially reflecting differences in homeostatic restoration versus hepatotoxic responses. Our study highlights the importance of considering early post-thawing metabolic dynamics in experimental design and offers insights for optimizing hepatocyte culture protocols to better replicate in vivo physiological conditions. Full article

Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver condition with potential progression to cirrhosis and impaired regeneration post-resection. A key mechanism underlying lipotoxicity is endoplasmic reticulum (ER) stress, particularly the activation of the unfolded protein response (UPR). This study investigates the interplay between lipid accumulation, endoplasmic reticulum (ER) stress, and cellular outcomes, focusing on the balance between autophagy and apoptosis. We cultured primary human hepatocytes (PHH) in a free fatty acid (FFA)-enriched medium for 120 h, assessing lipid accumulation, metabolism, and the expression of selected UPR markers. Additionally, we investigated the effects of lipid load on cell activity and growth in proliferating HepG2 cells. We observed that FFA uptake consistently induced ER stress, shifting cellular responses toward apoptosis under high lipid loads. Donor-specific differences were evident, particularly in lipid storage, excretion, and sensitivity to lipotoxicity. Some donors exhibited limited triglyceride (TAG) storage and excretion, leading to an excess of FFA whose metabolic fate remains unclear. Proliferation was more sensitive to lipid accumulation than overall cell activity, with even low FFA concentrations impairing growth, highlighting the vulnerability of regenerative processes to steatosis. The study elucidates how ER stress pathways, such as PERK-CHOP and IRE1α-JNK, are differentially activated in response to lipid overload, tipping the balance toward apoptosis in severe cases. The limited activation of repair mechanisms, such as autophagy, further emphasizes the critical role of ER stress in determining hepatocyte fate. The donor-dependent variability highlights the need for personalized strategies to mitigate lipotoxic effects and enhance liver regeneration in steatosis-related conditions. Full article

Background: Liver diseases are a global health concern. Many in vitro liver models utilize cryopreserved primary human hepatocytes (PHHs), which commonly undergo post-thaw processing through colloidal silica gradients to remove debris and enrich for a viable PHH population. Post-thaw processing effects on healthy PHHs are partially understood, but the consequences of applying disease-origin PHHs to post-thaw density gradient separation have not been described. Methods: Using the TruVivo^® system, diseased, type 2 diabetes mellitus (T2DM), and fibrotic PHHs were cultured for 14 days after initially being subjected to either low-density (permissive) or high-density (selective) gradients using Percoll-based thawing medium. Results: Changes in functionality, including albumin and urea secretion and CYP3A4 activity, were measured in diseased, T2DM, and fibrotic PHHs enriched in low Percoll compared to PHHs enriched in high Percoll. Lipogenesis increased in the PHHs enriched in low Percoll. Higher expression of CK18 and TGF-β, two fibrotic markers, and changes in expression of the macrophage markers CD68 and CD163 were also measured. Conclusions: The use of Percoll for the enrichment of PHHs post-thaw results in differences in attachment and functionality, along with changes in diseased phenotypes, in the TruVivo^® system. Full article

The liver is one of the key organs for exogenous and endogenous metabolism and is often a target for drug- and chemical-driven toxicity. A wide range of experimental approaches has been established to model and characterize the mechanisms of drug- and chemical-induced hepatotoxicity. A number of microfluidics-enabled in vitro models of the liver have been developed, but the unclear translatability of these platforms has hindered their adoption by the pharmaceutical industry; to achieve wide use for drug and chemical safety evaluation, demonstration of reproducibility and robustness under various contexts of use is required. One of these commercially available platforms is the PhysioMimix LC12, a microfluidic device where cells are seeded into a 3D scaffold that is continuously perfused with recirculating cell culture media to mimic liver sinusoids. Previous studies demonstrated this model’s functionality and potential applicability to preclinical drug development. However, to gain confidence in PhysioMimix LC12’s robustness and reproducibility, supplementary characterization steps are needed, including the assessment of various human hepatocyte sources, contribution of non-parenchymal cells (NPCs), and comparison to other models. In this study, we performed replicate studies averaging 14 days with either primary human hepatocytes (PHHs) or induced pluripotent stem cell (iPSC)-derived hepatocytes, with and without NPCs. Albumin and urea secretion, lactate dehydrogenase, CYP3A4 activity, and metabolism were evaluated to assess basal function and metabolic capacity. Model performance was characterized by different cell combinations under intra- and inter-experimental replication and compared to multi-well plates and other liver platforms. PhysioMimix LC12 demonstrated the highest metabolic function with PHHs, with or without THP-1 or Kupffer cells, for up to 10–14 days. iPSC-derived hepatocytes and PHHs co-cultured with additional NPCs demonstrated sub-optimal performance. Power analyses based on replicate experiments and different contexts of use will inform future study designs due to the limited throughput and high cell demand. Overall, this study describes a workflow for independent testing of a complex microphysiological system for specific contexts of use, which may increase end-user adoption in drug development. Full article

The hepatitis B virus (HBV) and hepatitis D virus (HDV) infections cause liver disease, including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV infection remains a major global health problem. In 2019, 296 million people were living with chronic hepatitis B and about 5% of them were co-infected with HDV. In vitro cell culture systems are instrumental in the development of therapeutic targets. Cell culture systems contribute to identifying molecular mechanisms for HBV and HDV propagation, finding drug targets for antiviral therapies, and testing antiviral agents. Current HBV therapeutics, such as nucleoside analogs, effectively suppress viral replication but are not curative. Additionally, no effective treatment for HDV infection is currently available. Therefore, there is an urgent need to develop therapies to treat both viral infections. A robust in vitro cell culture system supporting HBV and HDV infections (HBV/HDV) is a critical prerequisite to studying HBV/HDV pathogenesis, the complete life cycle of HBV/HDV infections, and consequently identifying new therapeutics. However, the lack of an efficient cell culture system hampers the development of novel antiviral strategies for HBV/HDV infections. In vitro cell culture models have evolved with significant improvements over several decades. Recently, the development of the HepG2-NTCP sec+ cell line, expressing the sodium taurocholate co-transporting polypeptide receptor (NTCP) and self-assembling co-cultured primary human hepatocytes (SACC-PHHs) has opened new perspectives for a better understanding of HBV and HDV lifecycles and the development of specific antiviral drug targets against HBV/HDV infections. We address various cell culture systems along with different cell lines and how these cell culture systems can be used to provide better tools for HBV and HDV studies. Full article

Non-alcoholic fatty liver disease (NAFLD) begins with lipid accumulation within hepatocytes, but the relative contributions of different macronutrients is still unclear. We investigated the impact of fatty acids, glucose and fructose on lipid accumulation in primary human hepatocytes (PHH) and three different cell lines: HepG2 (human hepatoblastoma–derived cell line), Huh7 (human hepatocellular carcinoma cell line) and McA-RH7777 (McA, rat hepatocellular carcinoma cell line). Cells were treated for 48 h with fatty acids (0 or 200 μM), glucose (5 mM or 11 mM) and fructose (0 mM, 2 mM or 8 mM). Lipid accumulation was measured via Nile Red staining. All cell types accumulated lipid in response to fatty acids (p < 0.001). PHH and McA, but not HepG2 or Huh7 cells, accumulated more lipid with 11 mM glucose plus fatty acids (p = 0.004, fatty acid × glucose interaction, for both), but only PHH increased lipid accumulation in response to fructose (p < 0.001). Considerable variation was observed between PHH cells from different individuals. Lipid accumulation in PHH was increased by insulin (p = 0.003) with inter-individual variability. Similarly, insulin increased lipid accumulation in both HepG2 and McA cells, with a bigger response in McA in the presence of fatty acids (p < 0.001 for fatty acid × insulin). McA were more insulin sensitive than either HepG2 or Huh7 cells in terms of AKT phosphorylation (p < 0.001 insulin × cell type interaction). Hence, glucose and fructose can contribute to the accumulation of lipid in PHH with considerable inter-individual variation, but hepatoma cell lines are not good models of PHH. Full article

Metabolic alterations in hepatocellular carcinoma (HCC) are fundamental for the development of diagnostic screening and therapeutic intervention since energy metabolism plays a central role in differentiated hepatocytes. In HCC research, hepatoma cell lines (HCLs) like HepG2 and Huh7 cells are still the gold standard. In this study, we characterized the metabolic profiles of primary human hepatoma cells (PHCs), HCLs and primary human hepatocytes (PHHs) to determine their differentiation states. PHCs and PHHs (HCC-PHHs) were isolated from surgical specimens of HCC patients and their energy metabolism was compared to PHHs from non-HCC patients and the HepG2 and Huh7 cells at different levels (transcript, protein, function). Our analyses showed successful isolation of PHCs with a purity of 50–73% (CK18+). The transcript data revealed that changes in mRNA expression levels had already occurred in HCC-PHHs. While many genes were overexpressed in PHCs and HCC-PHHs, the changes were mostly not translated to the protein level. Downregulated metabolic key players of PHCs revealed a correlation with malign transformation and were predominantly pronounced in multilocular HCC. Therefore, HCLs failed to reflect these expression patterns of PHCs at the transcript and protein levels. The metabolic characteristics of PHCs are closer to those of HCC-PHHs than to HCLs. This should be taken into account for future optimized tumor metabolism research. Full article

There are two main enzymes that convert tryptophan (Trp) to kynurenine (Kyn): tryptophan-2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Kyn accumulation can promote immunosuppression in certain cancers. In this study, we investigated Trp degradation to Kyn by IDO and TDO in primary human hepatocytes (PHH) and tumoral HepaRG cells. To quantify Trp-degradation and Kyn-accumulation, using reversed-phase high-pressure liquid chromatography, the levels of Trp and Kyn were determined in the culture media of PHH and HepaRG cells. The role of IDO in Trp metabolism was investigated by activating IDO with IFN-γ and inhibiting IDO with 1-methyl-tryptophan (1-DL-MT). The role of TDO was investigated using one of two TDO inhibitors: 680C91 or LM10. Real-time PCR was used to measure TDO and IDO expression. Trp was degraded in both PHH and HepaRG cells, but degradation was higher in PHH cells. However, Kyn accumulation was higher in the supernatants of HepaRG cells. Stimulating IDO with IFN-γ did not significantly affect Trp degradation and Kyn accumulation, even though it strongly upregulated IDO expression. Inhibiting IDO with 1-DL-MT also had no effect on Trp degradation. In contrast, inhibiting TDO with 680C91 or LM10 significantly reduced Trp degradation. The expression of TDO but not of IDO correlated positively with Kyn accumulation in the HepaRG cell culture media. Furthermore, TDO degraded L-Trp but not D-Trp in HepaRG cells. Kyn is the main metabolite of Trp degradation by TDO in HepaRG cells. The accumulation of Kyn in HepaRG cells could be a key mechanism for tumor immune resistance. Two TDO inhibitors, 680C91 and LM10, could be useful in immunotherapy for liver cancers. Full article

Non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in the liver, is the most common cause of liver diseases in Western countries. NAFLD is a major risk factor for developing hepatocellular carcinoma (HCC); however, in vitro evaluation of hepatic cancerogenesis fails due to a lack of liver models displaying a proliferation of hepatocytes. Originally designed to overcome primary human hepatocyte (PHH) shortages, upcyte hepatocytes were engineered to obtain continuous proliferation and, therefore, could be a suitable tool for HCC research. We generated upcyte hepatocytes, termed HepaFH3 cells, and compared their metabolic characteristics to HepG2 hepatoma cells and PHHs isolated from resected livers. For displaying NAFLD-related HCCs, we induced steatosis in all liver models. Lipid accumulation, lipotoxicity and energy metabolism were characterized using biochemical assays and Western blot analysis. We showed that proliferating HepaFH3 cells resemble HepG2, both showing a higher glucose uptake rate, lactate levels and metabolic rate compared to PHHs. Confluent HepaFH3 cells displayed some similarities to PHHs, including higher levels of the transaminases AST and ALT compared to proliferating HepaFH3 cells. We recommend proliferating HepaFH3 cells as a pre-malignant cellular model for HCC research, while confluent HepaFH3 cells could serve as PHH surrogates for energy metabolism studies. Full article