Co-culture Model Of Hepatocytes Research Articles

Assessment of covalent binding body burden in Hmrel human hepatocyte co-culture model for metabollically low turnover compounds

Assessment of covalent binding body burden in Hmrel human hepatocyte co-culture model for metabollically low turnover compounds

Application of Covalent Binding Body Burden in the HμREL Human Hepatocyte Coculture Model for Reactivity Risk Assessment of Metabolically Low Turnover Drugs.

The human hepatocyte suspension model has been a valuable tool to study covalent binding (CVB) for compounds that form reactive metabolites. However, accurately measuring CVB values with the suspension model becomes challenging for metabolically low turnover compounds. In this study, we evaluated the HμREL human hepatocyte coculture model relative to existing literature using human hepatocyte suspension for drugs of known drug-induced liver injury category. Our results indicate that this coculture model provides ample metabolic turnover to reproducibly measure CVB. It is sufficiently robust to apply a predefined 1 mg/day CVB body burden threshold for risk assessment to guide our discovery programs, allowing for expanded coverage to include metabolically low turnover compounds.

Adipose-Derived Mesenchymal Stromal/Stem Cell Line Prevents Hepatic Ischemia/Reperfusion Injury in Rats by Inhibiting Inflammasome Activation

Mesenchymal stromal/stem cells (MSCs) have shown potential in the treatment of degenerative diseases, including ischemia/reperfusion injury (IRI), which occurs during organ transplantation and represents the main cause of post-transplant graft dysfunction. However, MSCs have heterogeneous characteristics, and studies of MSCs therapy have shown a variety of outcomes. To establish a new effective MSCs therapy, we developed an adipose-derived mesenchymal stromal/stem cell line (ASCL) and compared its therapeutic effects on primary adipose-derived MSCs (ASCs) using a hepatocyte co-culture model of hypoxia/reoxygenation in vitro and a rat model of hepatic IRI in vivo. The results showed that both ASCL and ASCs protect against hypoxia by improving hepatocyte viability, inhibiting reactive oxygen species release, and upregulating transforming growth factor-β in vitro. In vivo, ASCL or ASCs were infused into the spleen 24 h before the induction of rat hepatic IRI. The results showed that ASCL significantly improved the survival outcomes compared with the control (normal saline infusion) with the significantly decreased serum levels of liver enzymes and less damage to liver tissues compared with ASCs. Both ASCL and ASCs suppressed NOD-like receptor family pyrin domain-containing 3 inflammasome activation and subsequently reduced the release of activated IL-1β and IL-18, which is considered an important mechanism underlying ASCL and ASCs infusion in hepatic IRI. In addition, ASCL can promote the release of interleukin-1 receptor antagonist, which was previously reported as a key factor in hampering the inflammatory cascade during hepatic IRI. Our results suggest ASCL as a new candidate for hepatic IRI treatment due to its relatively homogeneous characteristics.

Development of a co-culture model of mouse primary hepatocytes and splenocytes to evaluate xenobiotic genotoxicity using the medium-throughput Comet assay

To date, only a limited number of toxicological studies have focused on the establishment and validation of in vitro genotoxicity screening systems using primary hepatocytes, and the results of these studies have been inconsistent. Therefore, the aim of this study was to develop an effective co-culture model of mouse-derived primary hepatocytes and splenocytes for screening chemicals for genotoxicity using the medium-throughput Comet assay. This cocultured model was constructed and verified using known genotoxic and non-genotoxic compounds as positive and negative controls, respectively. Cytotoxicity was measured using Cell Counting Kit-8 and lactate dehydrogenase methods. DNA damage was detected using both alkaline and formamidopyrimidine DNA glycosylase (FPG) Comet assays. Compared with the controls, DNA strand breaks and FPG-sensitive sites showed significant concentration-dependent increases in genotoxic-agent-treated groups. In contrast, DNA damage remained unchanged in non-genotoxic-agent-treated groups. In addition, different types of genotoxic agents resulted in different time-dependent DNA lesions. Our results indicated that the % tail DNA indicating both DNA strand breaks and FPG-sensitive sites might be effective markers for predicting chemical-induced DNA damage and oxidative DNA damage using the cocultured model of hepatocytes and splenocytes. Collectively, these findings provide reliable experimental data for the establishment of in vitro genotoxicity screening methods.

P135 - Optimization of non-parenchymal and hepatocyte co-culture model

P135 - Optimization of non-parenchymal and hepatocyte co-culture model

P5 - Use of the HμREL® human hepatocyte co-culture model to determine intrinsic clearance and elucidate metabolic pathways of stable compounds

P5 - Use of the HμREL® human hepatocyte co-culture model to determine intrinsic clearance and elucidate metabolic pathways of stable compounds

Assessment of the Biotransformation of Low-Turnover Drugs in the HµREL Human Hepatocyte Coculture Model.

Metabolic profiles of four drugs possessing diverse metabolic pathways (timolol, meloxicam, linezolid, and XK469) were compared following incubations in both suspended cryopreserved human hepatocytes and the HμREL hepatocyte coculture model. In general, minimal metabolism was observed following 4-hour incubations in both suspended hepatocytes and the HμREL model, whereas incubations conducted up to 7 days in the HμREL coculture model resulted in more robust metabolic turnover. In the case of timolol, in vivo human data suggest that 22% of the dose is transformed via multistep oxidative opening of the morpholine moiety. Only the first-step oxidation was detected in suspended hepatocytes, whereas the relevant downstream metabolites were produced in the HµREL model. For meloxicam, both the hydroxymethyl and subsequent carboxylic acid metabolites were abundant following incubation in the HμREL model, while only a trace amount of the hydroxymethyl metabolite was observed in suspension. Similar to timolol, linezolid generated substantially higher levels of morpholine ring-opened carboxylic acid metabolites in the HμREL model. Finally, while the major aldehyde oxidase-mediated mono-oxidative metabolite of XK469 was minimally produced in hepatocyte suspension, the HμREL model robustly produced this metabolite, consistent with a pathway reported to account for 54% of the total urinary excretion in human. In addition, low-level taurine and glycine conjugates were identified in the HµREL model. In summary, continuous metabolite production was observed for up to 7 days of incubation in the HµREL model, covering cytochrome P450, aldehyde oxidase, and numerous conjugative pathways, while predominant metabolites correlated with relevant metabolites reported in human in vivo studies.

The Effect of Nitric Oxide on Ammonia Decomposition in Co-cultures of Hepatocytes and Hepatic Stellate Cells

Hepatic functions, such as albumin secretion and ammonia metabolism, are upregulated in response to hepatocyte growth factor (HGF) produced by hepatic stellate cells (HSC), as well as nitric oxide (NO) produced by endothelial cells under shear stress. However, the simultaneous effect of HSC and NO has not been previously investigated in a tri-co-culture model containing hepatocytes with HSC and endothelial cells under shear stress. We hypothesized that NO inhibits HGF production from HSC. To test this idea, we constructed a mono-culture model of hepatocytes and a co-culture model of hepatocytes and HSC and measured ammonia decomposition and HGF production in each model under NO load. Ammonia decomposition was significantly higher in the co-culture model under 0ppm NO load, but no significant increase was observed under NO load. In the co-culture model, HGF was produced at 1.0ng/mL under 0ppm NO load and 0.3ng/mL under NO load. Ammonia decomposition was increased by 1.0ng/mL HGF, but not by 0.3ng/mL HGF. These results indicated that NO inhibits HGF production from HSC; consequently, the effects of NO and co-culture with HSC cannot improve hepatic function simultaneously. Instead, the simultaneous effect of 1.0ng/mL HGF and NO may further enhance hepatic function in vitro.

The importance of the interaction between hepatocyte and hepatic stellate cells in fibrogenesis induced by fatty accumulation

Background & aimsNon-alcoholic fatty liver disease is characterized by an initial accumulation of triglycerides that can progress to non-alcoholic steatohepatitis, which can ultimately evolve to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells play a key role in liver fibrogenesis by an increased activation and an altered profile of genes involved in the turnover of extracellular matrix components. To reproduce in-vitro the functional cell connections observed in vivo it is essential to consider cell-to-cell proximity and interaction. The aim of this study was to determine the response to free fatty acids in a simultaneous co-culture model of hepatocytes and hepatic stellate cells. MethodsSimultaneous co-culture model and monoculture of each cell type (control) were exposed to FFA for 24 up to 144h. Quantification of steatosis; stellate cell activation; assessment of fibrogenic response; expression and activity of metalloproteinases as well as collagen biosynthesis were evaluated. ResultsFree fatty acids induced comparable steatosis in simultaneous co-culture and monoculture. However, the activation of the stellate cells assessed by alpha-smooth muscle actin expression is greater when cells were in close contact. Furthermore, a time-dependent increment of tissue inhibitor metalloproteinase-2 protein was observed, which was inversely correlated with protein expression and activity of matrix-metalloproteinases, suggesting enhanced collagen biosynthesis. This behavior was absent in cell monoculture. ConclusionsThese data indicate that cell-to-cell proximity between hepatocytes and stellate cells is necessary for the initiation of the fibrotic process.

A Micropatterned Hepatocyte Coculture Model for Assessment of Liver Toxicity Using High-Content Imaging Analysis

The current landscape of in vitro models used to identify drug- or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS. The coculture model consists of primary hepatocytes that are micropatterned to form a discrete microarchitecture or "hepatocyte islands" that are surrounded by supporting fibroblasts resulting in long-term viability and metabolic function of primary hepatocytes. Using multiple HCS image capture and image analysis strategies, we established methods to interrogate various morphometric parameters, such as size, shape, and intensity, at the island or single-cell level. We applied these approaches to identify subpopulations of both fibroblasts and hepatocytes that exhibited alterations in nuclear parameters, cell permeability, mitochondria function, and apoptosis using known reference control compounds and an eight-point dose curve. Subpopulation analysis with additional bioprobe sets can provide a powerful means of addressing differential cell and tissue susceptibilities during compound profiling. Our data show that the HepatoPac is amendable for HCS imaging applications and provides a unique approach for studying hepatotoxicity over prolonged periods of time.

Effect of Flow Load on Hepatic Function in Co-Culture of Hepatocytes with Hepatic Stellate Cells and Endothelial Cells: Relationship between Hepatic Function and Nitric Oxide Concentration <I>in vitro</I>

To date, no study has described the effects of media flow load on a co-culture model of hepatocytes (HC), hepatic stellate cells (HSC) and endothelial cells (EC). Furthermore, no research has been reported regarding the influence of nitric oxide (NO) concentration in such a co-culture model. Therefore, we developed co-culture models that include two or three of these cell types, and assayed their hepatic functions both in static culture and under flow load. We also measured the NO concentration in each models and inhibited NO production of cells. In static culture, the HC+HSC and HC+HSC+EC models demonstrated higher hepatic function than in the model containing HC alone. Under flow load, all models exhibited higher hepatic function than in static culture. The HC+HSC and HC+HSC+EC models under flow demonstrated the highest hepatic function observed under any condition. In almost all models, NO concentration exhibited the same tendency to increase along with hepatic function, and NO improved hepatic function in vitro without in HC+HSC model under flow load. Inhibition of NO production decreased small levels of hepatic function in HC+HSC and HC+HSC+EC models under flow load. We conclude that co-culture and flow load positively impact hepatic function, and that HSC and NO are related to improvements in hepatic function. Furthermore, we consider that the presence of HSC is responsible for other aspects of improvement in hepatic function.

A216 肝細胞共培養モデルにおける肝機能促進とNO濃度の関連性(A2-3 細胞のバイオメカニクス1)

A216 肝細胞共培養モデルにおける肝機能促進とNO濃度の関連性(A2-3 細胞のバイオメカニクス1)

Adipocyte Hypoxia Increases Hepatocyte Hepcidin Expression

Hepcidin plays a key role in regulating iron metabolism by blocking iron efflux from macrophages and enterocytes. Hepcidin is synthesized primarily in the liver, and its expression is increased by iron overload and inflammation. Obesity is associated with chronic inflammation as well as poor iron status. Central obesity causes adipocyte hypoxia resulting in chronic inflammation. Therefore, the objective of the present study was to determine if adipocyte hypoxia and associated inflammation signal hepatocyte hepcidin expression. The effect of adipocyte hypoxia on hepcidin expression was modeled using a 3T3-L1 adipocyte/Huh7 hepatocyte co-culture model. Adipocytes were cultured at either standard conditions (19% O2) or hypoxic conditions (1% O2). Compared to standard conditions, hypoxic 3T3-L1 cells had significantly higher IL-6 and leptin expression. Treatment of Huh7 cells with media from hypoxic or LPS-treated 3T3-L1 adipocytes significantly increased hepcidin promoter activity and mRNA compared to cells treated with normoxic 3T3-L1 media or control media. When the hepcidin STAT3 binding site was mutated, promoter activation by hypoxic media was abrogated. These data suggest that adipocyte hypoxia (a feature of central obesity) may increase hepcidin expression and plays a role in the association between obesity and poor iron status.

Leishmania chagasi: Cytotoxic effect of infected macrophages on parenchymal liver cells

Leishmania ( Leishmania) chagasi, the ethiological agent of New World visceral leishmaniasis, causes morphological and functional injury to the liver. To investigate the role of macrophage-released leishmanicidal factors in hepatocyte damage, we used a co-culture model of hepatocytes and L. chagasi promastigote-infected peritoneal macrophages obtained from C57BL/6 or BALB/c mice. C57BL/6 macrophages killed intracellular parasites more efficiently than BALB/c macrophages, leading to higher number of intracellular amastigotes in the BALB/c culture during the entire course of infection. Early TNF-α production led to macrophages activation resulting in parasite growth control. Hepatic transaminases and lactate dehydrogenase were present at high levels in the supernatants of both co-cultures; concurrently, parasites were eliminated from infected macrophages. Nitric oxide production was higher in C57BL/6 co-cultures than in BALB/c co-cultures. Inhibitors of the oxidative burst and secreted proteinases protected hepatocytes against toxicity, and treatment with an inducible nitric oxide synthase inhibitor fully impeded the enzyme release. Our data suggest that the intracellular cytotoxic effects elicited by macrophages for parasite destruction are directly associated with hepatocyte damage, and that nitric oxide plays a pivotal role in this phenomenon.

VEGF Is Upregulated in a Neuroblastoma and Hepatocyte Coculture Model

Background. We hypothesize that angiogenic factors are altered by the interaction between neuroblastoma cells and host tissues.Materials and methods. Human Chang hepatocytes and human neuroblastoma cells are cultured separately and in a noncontact, coculture system. Immunostaining for VEGF is performed on the cells. ELISA is used to detect vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and interleukin-8 in the conditioned media. Human umbilical vein endothelial cells (HUVEC) are cultured with standard medium (control) and hepatocyte, neuroblastoma, and coculture conditioned media. After 48 and 72 h, cells are counted to determine proliferation. Finally, VEGF-blocking antibody is added to the HUVEC cultures with the conditioned media.Results. VEGF is markedly elevated in the coculture medium compared to the media from hepatocytes or neuroblastoma grown alone [412.2 ± 52 vs 235 ± 35 or 74.5 ± 28.5 (pg/106 cells), P < 0.05]. Other growth factors are almost undetectable in any of the media. Immunostaining for VEGF in the cocultured hepatocytes is decreased by almost 50%, but VEGF immunostaining is increased fourfold in the cocultured neuroblastoma cells. A significant increase in cell proliferation is seen at both 48 and 72 h when HUVEC are cultured with the coculture media. Cell proliferation is blocked with the addition of anti-VEGF antibody.Conclusion. The interaction of neuroblastoma with hepatocytes results in an increased production of VEGF. It stimulates endothelial cell proliferation and may enhance the tumor's metastatic potential in an autocrine fashion.

Fas-Mediated Induction of Hepatocyte Apoptosis in a Neuroblastoma and Hepatocyte Coculture Model

Background.We have previously demonstrated an increase in hepatocyte apoptosis when they are cocultured with neuroblastoma cells. Death receptors in the tumor necrosis factor (TNF) family such as TNFR1 and Fas have been identified as regulators of apoptosis and may be responsible for the altered regulation of apoptosis seen in our coculture model. To evaluate the effects of released factors and remove the potential alterations induced by direct contact, a noncontact coculture system was used to study the interaction between hepatocytes and neuroblastoma cells.Methods.Human Chang hepatocytes (HC) were plated onto Falcon cell culture inserts with 0.45-μm pores in the permeable membrane. Human neuroblastoma cells (NB-IMR-32) were seeded into wells of the Falcon companion plate. After 24 h, inserts containing HC were placed into wells containing NB cells and incubated for 4 days. This provided a coculture environment without actual cellular contact. Immunohistochemical staining for TNFα, Fas, and Fas ligand (Fas-L) was performed. Apoptosis was detected via the TUNEL method. Images were analyzed with ImagePro-Plus. Statistical analyses were done with significance determined atP< 0.05.Results.Chang hepatocytes demonstrated a significant increase in the levels of TNF, Fas, and Fas-L when cocultured with neuroblastoma cells (P< 0.005). In addition, the cocultured hepatocytes had a 20-fold increase in the apoptotic rate (P< 0.001). Neuroblastoma cells had no demonstrable level of Fas or TNF when grown alone and in cocultures. Neuroblastoma cells that were grown alone had an elevated level of Fas-L, but this level diminished by 44% when cocultured with hepatocytes (P< 0.001).Conclusion.An upregulated TNF/Fas receptor–ligand system may be responsible for increased apoptosis in hepatocytes when cocultured with neuroblastoma. This upregulation may be due to release of neuroblastoma-derived Fas ligand into the media. Tumors may alter the regulation of apoptosis in surrounding tissues via the death receptors.

Induction of Apoptosis in a Neuroblastoma and Hepatocyte Coculture Model

Background.The dysregulation of apoptosis may alter the progression of tumor growth and explain the clinical dichotomy observed in children with neuroblastoma (NB). An overexpression of the bcl-2 proto-oncogene induces resistance to apoptosis and has been observed in unfavorable NB. We hypothesized that alterations in apoptosis may be a result of the interactions between NB and the tissues surrounding it.Materials and Methods.Human Chang liver cells (HCL, 104cells/cm2) were plated in two-chamber slides for 3 days. Human NB cells (105cells/cm2) were added to one of the chambers and incubated for 3 more days. Control NB were plated under identical conditions in its own medium and in the HCL medium with growth curves measured. DNA fragmentation was detected via the TUNEL method (TdT-mediated nick end-labeling) and bcl-2 expression was determined by immunostaining.Results.NB growth was unaltered by the change in medium. NB stained mildly positive for bcl-2 when plated alone but became markedly positive in coculture. Histologically, HCL and NB appeared healthy when plated alone, but a halo of apoptotic HCL was seen around NB in the coculture. When plated alone, both NB and HCL demonstrated minimal apoptotic activity as detected via the TUNEL method. In the coculture, a halo of HCL surrounding the NB exhibited markedly increased DNA fragmentation and this intensity diminished in cells distant from the NB.Conclusions.The regulation of apoptosis was altered in this coculture model of NB and HCL. HCL stimulated NB to overexpress bcl-2 and presumably become resistant to apoptosis. Conversely, NB induced the surrounding HCL to undergo apoptosis. The interaction between the local tissue and NB induced alterations in apoptosis in both cell types and resulted in a survival advantage for NB.