Search Results (27)

Changes in hepatic lipoprotein metabolism are responsible for the majority of metabolic dysfunction-associated disorders, including familial hypercholesterolemia (FH), metabolic syndrome (MetS), metabolic dysfunction-associated fatty liver disease (MAFLD), and age-related diseases such as atherosclerosis, a major health burden in modern society. This review aims to advance the understanding of state-of-the-art mechanistic concepts in lipoprotein metabolism, with a particular focus on lipoprotein uptake and secretion and their dysregulation in disease, and to provide a comprehensive overview of experimental models used to study these processes. Human lipoprotein research faces several challenges. First, significant differences in lipoprotein metabolism between humans and other species hinder the reliability of non-human model systems. Additionally, ethical constraints often limit studies on human lipoprotein metabolism using tracers. Lastly, while 2D hepatocyte cell culture systems are widely used, they are commonly of cancerous origins, limiting their physiological relevance and necessitating the use of more physiologically representative models. In this review, we will elaborate on key findings in lipoprotein metabolism, as well as limitations and challenges of currently available study tools, highlighting mechanistic insights throughout discussion of these models. These include human tracer studies, animal studies, 2D tissue culture-based systems derived from cancerous tissue as well as from induced pluripotent stem cells (iPSCs)/embryonic stem cells (ESCs). Finally, we will discuss precision-cut liver slices, liver-on-a-chip models, and, particularly, improved 3D models: (i) spheroids generated from either hepatoma cancer cell lines or primary human hepatocytes and (ii) organoids generated from liver tissues or iPSCs/ESCs. In the last section, we will explore future perspectives on liver-in-a-dish models in studying mechanisms of liver diseases, treatment options, and their applicability in precision medicine approaches. By comparing traditional and advanced models, this review will highlight the future directions of lipoprotein metabolism research, with a focus on the growing potential of 3D liver organoid models. Full article

Three-dimensional (3D) fish hepatocyte cultures are promising alternative models for replicating in vivo data. Few studies have attempted to characterise the structure and function of fish 3D liver models and illustrate their applicability. This study aimed to further characterise a previously established spheroid model obtained from juvenile brown trout (Salmo trutta) primary hepatocytes under estrogenic stimulation. The spheroids were exposed for six days to environmentally relevant concentrations of 17α-ethinylestradiol—EE2 (1–100 ng/L). The mRNA levels of peroxisome (catalase—Cat and urate oxidase—Uox), lipid metabolism (acyl-CoA long chain synthetase 1—Acsl1, apolipoprotein AI—ApoAI, and fatty acid binding protein 1—Fabp1), and estrogen-related (estrogen receptor α—ERα, estrogen receptor β—ERβ, vitellogenin A—VtgA, zona pellucida glycoprotein 2.5—ZP2.5, and zona pellucida glycoprotein 3a.2—ZP3a.2) target genes were evaluated by quantitative real-time polymerase chain reaction. Immunohistochemistry was used to assess Vtg and ZP protein expressions. At the highest EE2 concentration, VtgA and ZP2.5 genes were significantly upregulated. The remaining target genes were not significantly altered by EE2. Vtg and ZP immunostaining was consistently increased in spheroids exposed to 50 and 100 ng/L of EE2, whereas lower EE2 levels resulted in a weaker signal. EE2 did not induce significant changes in the spheroids’ viability and morphological parameters. This study identified EE2 effects at environmentally relevant doses in trout liver spheroids, indicating its usefulness as a proxy for in vivo impacts of xenoestrogens. Full article

Background: Three-dimensional cellular models provide a more comprehensive representation of in vivo cell properties, encompassing physiological characteristics and drug susceptibility. Methods: Primary hepatocytes were seeded in ultra-low attachment plates to form spheroids, with or without tumoral cells. Spheroid structure, cell proliferation, and apoptosis were analyzed using histological staining techniques. In addition, extracellular vesicles were isolated from conditioned media by differential ultracentrifugation. Spheroids were exposed to cytotoxic drugs, and both spheroid growth and cell death were measured by microscopic imaging and flow cytometry with vital staining, respectively. Results: Concerning spheroid structure, an active outer layer forms a boundary with the media, while the inner core comprises a mass of cell debris. Hepatocyte-formed spheroids release vesicles into the extracellular media, and a decrease in the concentration of vesicles in the culture media can be observed over time. When co-cultured with tumoral cells, a distinct distribution pattern emerges over the primary hepatocytes, resulting in different spheroid conformations. Tumoral cell growth was compromised upon antitumoral drug challenges. Conclusions: Treatment of mixed spheroids with different cytotoxic drugs enables the characterization of drug effects on both hepatocytes and tumoral cells, determining drug specificity effects on these cell types. Full article

Adult stem cells (ASCs) can be cultured with difficulty from most tissues, often requiring chemical or transgenic modification to achieve adequate quantities. We show here that mouse primary fibroblasts, grown in suspension, change from the elongated and flattened morphology observed under standard adherent culture conditions of generating rounded cells with large nuclei and scant cytoplasm and expressing the mesenchymal stem cell (MSC) marker (Sca1; Ly6A) within 24 h. Based on this initial observation, we describe here a suspension culture method that, irrespective of the lineage used, mouse fibroblast or primary human somatic cells (fibroblasts, hepatocytes and keratinocytes), is capable of generating a high yield of cells in spheroid form which display the expression of ASC surface markers, circumventing the anoikis which often occurs at this stage. Moreover, mouse fibroblast-derived spheroids can be differentiated into adipogenic and osteogenic lineages. An analysis of single-cell RNA sequence data in mouse fibroblasts identified eight distinct cell clusters with one in particular comprising approximately 10% of the cells showing high levels of proliferative capacity expressing high levels of genes related to MSCs and self-renewal as well as the extracellular matrix (ECM). We believe the rapid, high-yield generation of proliferative, multi-potent ASC-like cells via the process we term suspension-induced stem cell transition (SIST) could have significant implications for regenerative medicine. Full article

In this study, we generated hepatocyte organoids (HOs) using frozen-thawed primary hepatocytes (PHs) within a three-dimensional (3D) Matrigel dome culture in a porcine model. Previously studied hepatocyte organoid analogs, spheroids, or hepatocyte aggregates created using PHs in 3D culture systems have limitations in their in vitro lifespans. By co-culturing adipose tissue-derived mesenchymal stem cells (A-MSCs) with HOs within a 3D Matrigel dome culture, we achieved a 3.5-fold increase in the in vitro lifespan and enhanced liver function compared to a conventional two-dimensional (2D) monolayer culture, i.e., more than twice that of the HO group cultured alone, reaching up to 126 d. Although PHs were used to generate HOs, we identified markers associated with cholangiocyte organoids such as cytokeratin 19 and epithelial cellular adhesion molecule (EPCAM). Co-culturing A-MSCs with HOs increased the secretion of albumin and urea and glucose consumption compared to HOs cultured alone. After more than 100 d, we observed the upregulation of tumor protein P53 (TP53)-P21 and downregulation of EPCAM, albumin (ALB), and cytochrome P450 family 3 subfamily A member 29 (CYP3A29). Therefore, HOs with function and longevity improved through co-culturing with A-MSCs can be used to create large-scale human hepatotoxicity testing models and precise livestock nutrition assessment tools. Full article

Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate. Full article

Mammal hepatocyte spheroids have been investigated as alternative experimental models in several contexts, since three-dimensional (3D) systems have shown the potential to mimic in vivo scenarios. The description of fish hepatocyte 3D models is still minimal. This study intends to further characterize brown trout primary hepatocyte spheroids at distinct time points up to 25 days in culture. Viability, biometry, histomorphology, and basal expression of a selection of genes (metabolism and detoxification, efflux transport, and estrogenic signalling) were considered. The gene expression of whole liver samples from the same fish donor were evaluated concurrently. After 12 days in culture, the hepatocyte spheroids exhibited biometric and morphological stability. From the 12th to the 20th day in culture, the basal expression levels for most of the selected genes did not vary. The targeted mRNA levels were higher in brown trout liver samples compared to hepatocyte spheroids. Despite that, data supported that this model resembles some in vivo features. As an experimental alternative model, it showed potential to be used in a stable time window that can be exploited for exposure tests to different xenobiotics, namely, estrogenic compounds. Full article

To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients. Full article

Establishing a rapid in vitro evaluation system for drug screening is essential for the development of new drugs. To reproduce tissues/organs with functions closer to living organisms, in vitro three-dimensional (3D) culture evaluation using microfabrication technology has been reported in recent years. Culture on patterned substrates with controlled hydrophilic and hydrophobic regions (Cell-able^TM) can create 3D liver models (miniature livers) with liver-specific Disse luminal structures and functions. MRI contrast agents are widely used as safe and minimally invasive diagnostic methods. We focused on anionic polysaccharide magnetic iron oxide nanoparticles (Resovist^®) and synthesized the four types of nanoparticle derivatives with different properties. Cationic nanoparticles (TMADM) can be used to label target cells in a short time and have been successfully visualized in vivo. In this study, we examined the morphology of various nanoparticles. The morphology of various nanoparticles showed relatively smooth-edged spherical shapes. As 3D liver models, we prepared primary hepatocyte–endothelial cell heterospheroids. The toxicity, CYP3A, and albumin secretory capacity were evaluated in the heterospheroids labeled with various nanoparticles. As the culture period progressed, the heterospheroids labeled with anionic and cationic nanoparticles showed lower liver function than non-labeled heterospheroids. In the future, there is a need to improve the method of creation of artificial 3D liver or to design a low-invasive MRI contrast agent to label the artificial 3D liver. Full article

Yellow fever virus (YFV) is a potentially lethal, zoonotic, blood-borne flavivirus transmitted to humans and non-human primates by mosquitoes. Owing to multiple deadly epidemics, the WHO classifies YFV as a “high impact, high threat disease” with resurgent epidemic potential. At present, there are no approved antiviral therapies to combat YFV infection. Herein we report on 2′-halogen-modified nucleoside analogs as potential anti-YFV agents. Of 11 compounds evaluated, three showed great promise with low toxicity, high intracellular metabolism into the active nucleoside triphosphate form, and sub-micromolar anti-YFV activity. Notably, we investigated a 2′-fluoro,2′-bromouridine phosphate prodrug (C9), a known anti-HCV agent with good stability in human blood and favorable metabolism. Predictive modeling revealed that C9 could readily bind the active site of the YFV RdRp, conferring its anti-YFV activity. C9 displayed potent anti-YFV activity in primary human macrophages, 3D hepatocyte spheroids, and in mice. In an A129 murine model, shortly after infection, C9 significantly reduced YFV replication and protected against YFV-induced liver inflammation and pathology with no adverse effects. Collectively, this work identifies a potent new anti-YFV agent with strong therapeutic promise. Full article

Background: Bergamot polyphenolic fraction (PF) extract exerts a beneficial against liver steatosis. However, the fundamental processes underlying this beneficial effect of bergamot PF remain elusive. In this work, we examined the effect of bergamot PF extract on 2D and 3D hepatocyte cultures. Material and Methods: We evaluated the effect of bergamot PF in 2D and 3D cultures from rat, human hepatoma cells, and human primary hepatocytes. Results: In 2D cell culture, we demonstrated that incubation with bergamot PF decreases intracellular lipid content and is associated with an increase in expression levels of ß-oxidation genes (Acox1, Pparα, and Ucp2) and lipophagy (Atg7). Moreover, we confirm this effect on 3D spheroids and organoids. Conclusion: Incubation with bergamot PF reduces intracellular lipid neutral fat potentially by increasing intracellular pathways related to beta-oxidation. Full article

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source to replace human primary hepatocytes. We previously developed genetically modified murine hepatoma cell lines with inducible enhanced liver functions, in which eight liver-enriched transcription factor (LETF) genes were introduced into hepatoma cells as inducible transgene expression cassettes. Here, we establish a human hepatoma cell line with heat-inducible liver functions using HepG2 cells. The genetically modified hepatoma cells, designated HepG2/8F_HS, actively proliferated under normal culture conditions and, therefore, can be easily prepared in large quantities. When the expression of LETFs was induced by heat treatment at 43 °C for 30 min, cells ceased proliferation and demonstrated enhanced liver functions. Furthermore, three-dimensional spheroid cultures of HepG2/8F_HS cells showed a further increase in liver functions upon heat treatment. Comprehensive transcriptome analysis using DNA microarrays revealed that HepG2/8F_HS cells had enhanced overall expression of many liver function-related genes following heat treatment. HepG2/8F_HS cells could be useful as a new cell source for pharmacological studies and for constructing bioartificial liver systems. Full article

Hepatocellular carcinoma (HCC) is the most dominant primary liver cancer, which can be caused by chronic hepatitis virus infections and other environmental factors. Resection, liver transplantation, and local ablation are only a few of the highly effective and curative procedures presently accessible. However, other complementary treatments can reduce cancer treatment side effects. In this present work, we evaluated the activity of Moroccan scorpion venom Buthus occitanus and its fractions obtained by chromatography gel filtration against HCC cells using a 3D cell culture model. The venom was fractionated by gel filtration chromatography, each fraction and the crude venom was tested on normal hepatocytes (Fa2N-4 cells). Additionally, the fractions and the crude venom were tested on MCTSs (multicellular tumor spheroids), and this latter was generated by cultivate Huh7.5 cancer cell line with WI38 cells, LX2 cells, and human endothelial cells (HUVEC). Our results indicate that Buthus occitanus venom toxin has no cytotoxic effects on normal hepatocytes. Moreover, it is reported that F3 fraction could significantly inhibit the MCTS cells. Other Protein Separation Techniques (High-performance liquid chromatography) are needed in order to identify the most active molecule. Full article

Background: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). Methods: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. Results: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. Conclusions: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications. Full article

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro. Full article