Hepatocyte Monocultures Research Articles

Comparison of Rat Hepatocyte 2D-Monocultures and Hepatocytes Non-Parenchymal Cell Co-Cultures for Assessing Chemical Toxicity.

Liver responses are the most common endpoints used as the basis for setting exposure standards. Liver hepatocytes play a vital role in biotransformation of xenobiotics, but non-parenchymal cells (NPCs) in the liver are also involved in certain liver responses. Development of in vitro systems that more faithfully capture liver responses to reduce reliance on animals is a major focus of New Approach Methodology (NAMs). Since rodent regulatory studies are frequently the sole source safety assessment data, mode-of-action data, and used for risk assessments, in vitro rodent models that reflect in vivo responses need to be developed to reduce reliance on animal models. In the work presented in this paper, we developed a 2-D hepatocyte monoculture and 2-D liver cell co-culture system using rat liver cells. These models were assessed for conditions for short-term stability of the cultures and phenotypic and transcriptomic responses of 2 prototypic hepatotoxicants compounds - acetaminophen and phenobarbital. The optimized multi-cellular 2-D culture required use of freshly prepared hepatocytes and NPCs from a single rat, a 3:1 ratio of hepatocytes to NPCs and growth medium using 50% Complete Williams E medium (WEM) and 50% Endothelial Cell Medium (ECM). The transcriptomic responses of the 2 model systems to PB were compared to previous studies from TG-Gates on the gene expression changes in intact rats and the co-culture model responses were more representative of the in vivo responses. Transcriptomic read-outs promise to move beyond conventional phenotypic evaluations with these in vitro NAMs and provide insights about modes of action.

The Effects of Matriptase Inhibition on the Inflammatory and Redox Homeostasis of Chicken Hepatic Cell Culture Models.

The function of the transmembrane serine protease matriptase is well described in mammals, but it has not been elucidated in avian species yet. Hence, the aim of the present study was to assess the effects of the 3-amidinophenylalanine (3-AphA)-type matriptase inhibitors MI432 and MI460 on the inflammatory and oxidative state of chicken primary hepatocyte mono-cultures and hepatocyte–nonparenchymal cell co-cultures, the latter serving as a proper model of hepatic inflammation in birds. Cell cultures were exposed to MI432 and MI460 for 4 and 24 h at 10, 25, and 50 µM concentrations, and thereafter the cellular metabolic activity, extracellular interleukin (IL-)6, IL-8, H2O2 and malondialdehyde concentrations were monitored. Both inhibitors caused a transient moderate reduction in the metabolic activity following 4 h exposure, which was restored after 24 h, reflecting the fast hepatic adaptation potential to matriptase inhibitor administration. Furthermore, MI432 triggered an intense elevation in the cellular proinflammatory IL-6 and IL-8 production after both incubation times in all concentrations, which was not coupled to enhanced oxidative stress and lipid peroxidation based on unchanged H2O2 production, malondialdehyde levels and glutathione peroxidase activity. These data suggest that physiological matriptase activities might have a key function in retaining the metabolic and inflammatory homeostasis of the liver in chicken, without being a major modulator of the hepatocellular redox state.

ERRATUM.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

Effects of Acute Heat Stress on a Newly Established Chicken Hepatocyte-Nonparenchymal Cell Co-Culture Model.

Simple SummaryAmong many environmental stress factors, heat stress can intensely deteriorate animal health, welfare, and productivity in poultry farming. In the present study, a novel cell culture comprised of different liver cell types from chickens was established to serve as a model for studying the effects of acute heat stress. Short, 1 h heat exposure strongly affected liver cells by increasing metabolism, triggering oxidative stress, and decreasing the generation of certain mediatory molecules of the cellular stress response. However, these alterations were normalized after 2 h of heat stress, suggesting a fast adaptation of liver cells. The results of this study underline the impact of short-term heat stress as a potential harmful factor affecting liver function in chickens.Heat stress is one of the most important issues in broiler flocks impairing animal health and productivity. On a cellular level, excess heat exposure can trigger heat shock response acting for the restoration of cell homeostasis by several mechanisms, such as affecting heat shock protein synthesis, redox homeostasis and pro-inflammatory cytokine production. The major aim of this study was to establish a novel avian hepatocyte—nonparenchymal cell co-culture as a model for investigating the cellular effects of heat stress and its interaction with inflammation in chicken liver. Cell fractions were isolated by differential centrifugation from a freshly perfused chicken liver, and hepatocyte mono-cultures as well as hepatocyte–nonparenchymal cell co-cultures (with cell ratio 6:1, hepatocytes to nonparenchymal cells, mimicking a milder hepatic inflammation) were prepared. Isolated and cultured cells were characterized by flow cytometry and immunocytochemistry applying hepatocyte- and macrophage-specific antibodies. Confluent cell cultures were exposed to 43 °C temperature for 1 or 2 h, while controls were cultured at 38.5 °C. The metabolic activity, LDH enzyme activity, reactive oxygen species (H2O2) production, extracellular concentration of heat shock protein 70 (HSP70), and that of the pro-inflammatory cytokines interleukin (IL-)6 and IL-8 were assessed. Shorter heat stress applied for 1 h could strongly influence liver cell function by significantly increasing catabolic metabolism and extracellular H2O2 release, and by significantly decreasing HSP70, IL-6, and IL-8 production on both cell culture models. However, all these alterations were restored after 2 h heat exposure, indicating a fast recovery of liver cells. Hepatocyte mono-cultures and hepatocyte—nonparenchymal cell co-cultures responded to heat stress in a similar manner, but the higher metabolic rate of co-cultured cells may have contributed to a better capability of inflamed liver cells for accommodation to stress conditions. In conclusion, the established new primary cell culture models provide suitable tools for studying the hepatic inflammatory and stress response. The results of this study highlight the impact of short-term heat stress on the liver in chickens, underline the mediatory role of oxidative stress in acute stress response, and suggest a fast cellular adaptation potential in liver cells.

Cellular Effects of T-2 Toxin on Primary Hepatic Cell Culture Models of Chickens.

Trichothecene mycotoxins such as T-2 toxin cause severe problems for agriculture, as well as for veterinary medicine. As liver is one of the key organs in metabolism, the main aim of our study was to investigate the immunomodulatory and cytotoxic effects of T-2 toxin, using primary hepatocyte mono-culture and hepatocyte—nonparenchymal cell (predominantly Kupffer cell) co-culture models of chicken. Cultures were exposed to 10 (T10 group), 100 (T100 group) and 1000 (T1000 group) nmol/L T-2 toxin treatment for 8 or 24 h. Alterations of cellular metabolic activity, the production of reactive oxygen species (extracellular H2O2), heat shock protein 70 (HSP70), and the concentration of different inflammatory cytokines such as interleukin (IL-)6 and IL-8 were investigated. Metabolic activity was intensely decreased by T-2 toxin administration in all of the cell culture models, in every applied concentration and incubation time. Concentrations of HSP70 and IL-8 were significantly increased in hepatocyte mono-cultures exposed to higher T-2 toxin levels (both in T100 and T1000 groups for HSP70 and in T1000 group for IL-8, respectively) compared to controls after 24 h incubation. Similarly, IL-6 levels were also significantly elevated in the T100 and T1000 groups in both of mono- and co-cultures, but only after 8 h of incubation time. In spite of the general harmful effects of T-2 toxin treatment, no significant differences were observed on reactive oxygen species production. Furthermore, the two cell culture models showed different levels of H2O2, HSP70, and IL-8 concentrations independently of T-2 toxin supplementation. In conclusion, the established primary cell cultures derived from chicken proved to be proper models to study the specific molecular effects caused by T-2 toxin. Metabolic activity and immune status of the different examined cell cultures were intensively affected; however, no changes were found in H2O2 levels.

Liver slice culture as a model for lipid metabolism in fish.

Hepatic lipid metabolism is traditionally investigated in vitro using hepatocyte monocultures lacking the complex three-dimensional structure and interacting cell types essential liver function. Precision cut liver slice (PCLS) culture represents an alternative in vitro system, which benefits from retention of tissue architecture. Here, we present the first comprehensive evaluation of the PCLS method in fish (Atlantic salmon, Salmo salar L.) and validate it in the context of lipid metabolism using feeding trials, extensive transcriptomic data, and fatty acid measurements. We observe an initial period of post-slicing global transcriptome adjustment, which plateaued after 3 days in major metabolic pathways and stabilized through 9 days. PCLS fed alpha-linolenic acid (ALA) and insulin responded in a liver-like manner, increasing lipid biosynthesis gene expression. We identify interactions between insulin and ALA, where two PUFA biosynthesis genes that were induced by insulin or ALA alone, were highly down-regulated when insulin and ALA were combined. We also find that transcriptomic profiles of liver slices are exceedingly more similar to whole liver than hepatocyte monocultures, both for lipid metabolism and liver marker genes. PCLS culture opens new avenues for high throughput experimentation on the effect of “novel feed composition” and represent a promising new strategy for studying genotype-specific molecular features of metabolism.

Long-Term Engineered Cultures of Primary Mouse Hepatocytes for Strain and Species Comparison Studies During Drug Development.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

TGFβ1-mediated suppression of cytochrome P450(CYP) induction responses in rat hepatocyte-fibroblast co-cultures

Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver specific functions and maintaining hepatic polarity. However, the utility of hepatocyte co-culture models for studies, such as drug-drug interaction studies, has not been completely elucidated. In this study the induction of Cyp1a2, Cyp2b1/2, and Cyp3a2, the three major cytochrome P450 (CYP) isoforms in the rat liver, was evaluated in randomly mixed co-cultures and micropatterned co-cultures. We found that in both co-culture configurations, the drug-induced Cyp1a2, Cyp2b1/2, Cyp3a2 mRNA and activity were suppressed relative to those in monocultured hepatocytes. Further, we observed a significant increase in TGFβ1 production in the co-cultures. Addition of 100 pg/ml TGFβ1 to hepatocyte monocultures resulted in the suppression of Cyp1a2, Cyp2b1/2, and Cyp3a2 induction. These findings implicate TGFβ1 as one of the important factors impairing drug induced CYP induction in co-cultures and suggests that caution needs to be exercised in the use of hepatocyte-fibroblast co-cultures for CYP induction studies.

A polyelectrolyte multilayer platform for investigating growth factor delivery modes in human liver cultures.

Polyelectrolyte multilayers (PEMs) of chitosan and heparin are useful for mimicking growth factor (GF) binding to extracellular matrix (ECM) as in vivo. Here, we developed a PEM platform for delivering bound/adsorbed GFs to monocultures of primary human hepatocytes (PHHs) and PHH/non-parenchymal cell (NPC) co-cultures, which are useful for drug development and regenerative medicine. The effects of ECM protein coating (collagen I, fibronectin, and Matrigel®) and terminal PEM layer on PHH attachment/functions were determined. Then, heparin-terminated/fibronectin-coated PEMs were used to deliver varying concentrations of an adsorbed model GF, transforming growth factor β (TGFβ), to PHH monocultures while using soluble TGFβ delivery via culture medium as the conventional control. Soluble TGFβ delivery caused a severe, monotonic, and sustained downregulation of all PHH functions measured (albumin and urea secretions, cytochrome-P450 2A6 and 3A4 enzyme activities), whereas adsorbed TGFβ delivery caused transient upregulation of 3 out of 4 functions. Finally, functionally stable co-cultures of PHHs and 3T3-J2 murine embryonic fibroblasts were created on the heparin-terminated/fibronectin-coated PEMs modified with adsorbed TGFβ to elucidate similarities and differences in functional response relative to the monocultures. In conclusion, chitosan-heparin PEMs constitute a robust platform for investigating the effects of GF delivery modes on PHH monocultures and PHH/NPC co-cultures. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 971-984, 2018.

Metabolic Oscillations in Co-Cultures of Hepatocytes and Mesenchymal Stem Cells: Effects of Seeding Arrangement and Culture Mixing.

In vitro assembly of functional liver tissue is a prerequisite for the transplantation of tissue-engineered livers. There is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability. Recent reports have demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) can provide a number of cues promoting hepatocyte growth and development. In this study, the effects of BM-MSCs co-culture on hepatocyte metabolism were evaluated as a function of scaffold seeding arrangement. BM-MSCs were co-cultured with hepatocytes in porous chitosan-heparin scaffolds using several seeding arrangements. The seeded scaffolds were subjected to orbital shaking to enhance mass transfer. BM-MSC-hepatocyte co-cultures exhibited higher rates of hepatocyte-specific functions, compared to hepatocyte-only cultures, regardless of the seeding arrangement. Cells formed smaller-compact spheroids in the heterotypic systems compared to mono-cultures of hepatocytes only. The spheroids exhibited reduction in size with time in all conditions except for the condition where BM-MSCs were seeded one day after seeding hepatocytes. In this condition, spheroids increased in size due to BM-MSC proliferation. Spheroid size reduction was hypothesized to be the result of cyclic shear stresses generated by the orbital shaking. Furthermore, results suggested that BM-MSC seeding onto preformed hepatocyte spheroids provide a degree of shear-protection and trophic stimuli. Overall, the results indicate that co-culturing hepatocytes with BM-MSCs enhanced their metabolic functions for the first week of culture. J. Cell. Biochem. 118: 3003-3015, 2017. © 2017 Wiley Periodicals, Inc.

Long-term liver-specific functions of hepatocytes in electrospun chitosan nanofiber scaffolds coated with fibronectin.

In this study, a new 3D liver model was developed using biomimetic nanofiber scaffolds and co-culture system consisting of hepatocytes and fibroblasts for the maintenance of long-term liver functions. The chitosan nanofiber scaffolds were fabricated by the electrospinning technique. To enhance cellular adhesion and spreading, the surfaces of the chitosan scaffolds were coated with fibronectin (FN) by adsorption and evaluated for various cell types. Cellular phenotype, protein expression, and liver-specific functions were extensively characterized by immunofluorescent and histochemical stainings, albumin enzyme-linked immunosorbent assay and Cytochrome p450 detoxification assays, and scanning electron microscopy. The electrospun chitosan scaffolds exhibited a highly porous and randomly oriented nanofibrous structure. The FN coating on the surface of the chitosan nanofibers significantly enhanced cell attachment and spreading, as expected, as surface modification with this cell adhesion molecule on the chitosan surface is important for focal adhesion formation and integrin binding. Comparison of hepatocyte mono-cultures and co-cultures in 3D culture systems indicated that the hepatocytes in co-cultures formed colonies and maintained their morphologies and functions for prolonged periods of time. The 3D liver tissue model developed in this study will provide useful tools toward the development of engineered liver tissues for drug screening and tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2119-2128, 2017.

Metabolomics of an in vitro liver model containing primary hepatocytes assembling around an endothelial cell network: comparative study on the metabolic stability and the effect of acetaminophen treatment.

Recently, a novel culture system consisting of primary hepatocytes structured over a network of endothelial cells on the Engelbreth-Holm-Swarm (EHS) gel has been reported. This in vitro liver model on the EHS gel (IVLEHS) has been shown to maintain the expression of hepatic genes and their functional activity. Moreover, the IVLEHS was more sensitive to xenobiotics than hepatocyte monocultures, suggesting the potential utility of this culture system for compound hepatotoxicity screening. However, the effect of this three-dimensional structure formation on the cellular metabolic profile of hepatocytes in the IVLEHS is not well understood. To address this concern, we performed metabolome analysis using capillary electrophoresis-time of flight mass spectrometry. Between the IVLEHS and mono-cultured hepatocytes on the EHS gel, there was no significant difference in the levels of metabolites of the urea cycle and the tricarboxylic acid cycle, essential amino acids, and adenylate energy charge (AEC) which is an important indicator of cellular energy status. On the other hand, acetaminophen-dependent decrease of the AEC in the IVLEHS was greater than that in the monoculture, suggesting the higher sensitivity of IVLEHS to acetaminophen-induced hepatotoxicity which is caused by metabolic activation of this drug. Further analysis showed that the levels of taurocholate, one of the major conjugated bile acids, were higher in the IVLEHS than in the monoculture. Considering that the construction of the IVLEHS did not seem to disturb the major cellular metabolism, our findings would strengthen the concept that IVLEHS would have beneficial effects on the maintenance of hepatic functions.

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca2+ oscillation and sustained Ca2+ signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.

Evaluation of the Interplay between Uptake Transport and CYP3A4 Induction in Micropatterned Cocultured Hepatocytes.

Previously we assessed the inductive response of prototypical inducers in hepatocyte monocultures and the long-term coculture model HepatoPac using cryopreserved hepatocytes from the same donors. We noted that the rifampicin EC50 generated using the HepatoPac model corresponded better to the EC50 based on clinical data compared with data generated in the monoculture system. We postulated that there may be differences in the functioning of uptake transporters between the two systems that may have led to the EC50 difference. In this study, we characterized the functional activity of multiple uptake transporters in the two systems using cryopreserved hepatocytes from the same donors. Our data suggest that uptake transporter activity is higher in HepatoPac compared with the monoculture system. As a follow up to this study, we measured the intracellular concentrations of rifampicin and bosentan, which are known substrates of uptake transporters; we observed significantly higher intracellular concentrations of both compounds in HepatoPac relative to the monoculture system. This finding equated to lower cytochrome P450 isoform 3A4 (CYP3A4) EC50 values in the HepatoPac system compared with the monoculture system for both mRNA and activity. In parallel, no significant EC50 shift was observed for carbamazepine and phenytoin, which are not known to be substrates of uptake transporters. Our data suggest that next generation liver models such as HepatoPac may be a useful in vitro tool to quantitatively predict drug-drug interactions when it is known that the perpetrator is also a substrate of drug transporters.

Porcine hepatocyte-Kupffer cell co-culture as an invitro model for testing the efficacy of anti-inflammatory substances.

As Kupffer cells are highly involved in the regulation of hepatic inflammatory response, the main goal of this study was to improve and to characterize a hepatocyte-Kupffer cell co-culture of pig origin for modelling endotoxin-induced hepatic inflammation and for testing the efficacy of potential anti-inflammatory substances. This monolayer co-culture was prepared from primary isolated swine hepatocytes and Kupffer cells in the ratio of 6:1 and 2:1, mimicking different states of liver inflammation. The prepared cell cultures were characterized by immunohistochemical CD-68 detection. Lipopolysaccharide (LPS) challenge of both co-cultures resulted in elevated interleukin-8 (IL-8) and that of 6:1 co-cultures in increased IL-6 production with a higher extent than on hepatocyte monocultures, justifying the key role of Kupffer cells in pro-inflammatory cytokine production. LPS-induced IL-8 production was successfully attenuated by concomitant application of both sodium butyrate and terpinen-4-ol on hepatocyte monocultures, but not on co-cultures, demonstrating the importance of the presence of Kupffer cells in cell cultures as inflammatory models. Based on these initial data, the applied porcine primary hepatocyte-Kupffer cell co-culture is suggested to be a proper tool for invitro investigations on liver physiology and hepatic inflammation in pigs and can be used as a useful model mimicking invivo conditions in veterinary research.

Comparative study of CYP2B1/2 induction and the transport of bilirubin and taurocholate in rat hepatocyte-mono- and hepatocyte-Kupffer cell co-cultures

IntroductionHepatocyte-Kupffer cell (KC) co-cultures represent a promising approach for in vitro modeling of complex interactions between parenchymal and non-parenchymal cells in the liver, responsible for drug-induced liver injury (DILI). In this study we aimed to compare hepatocyte monocultures with hepatocyte-KC co-cultures regarding some basic liver functions associated with the chemical defense system. These pathways involve transporters and enzymes the function of which is highly sensitive towards hepatotoxic events. MethodsCYP2B1/2 induction and the biliary and sinusoidal elimination of bilirubin (B) and taurocholate (TC) were studied in rat hepatocyte sandwich cultures compared with rat hepatocyte-KC sandwich co-cultures of 1:0, 6:1, 2:1 and 1:1 cell combinations representing the physiologic and pathologic conditions of the liver. ResultsKCs decreased phenobarbital inducibility of CYP2B1/2 in a cell ratio dependent manner and activation of KCs by lipopolisacharide (LPS) amplified this effect. Similarly, KCs decreased the transport of B and its glucuronides (BG) in both sinusoidal and canalicular directions resulting in its intracellular accumulation. In contrast, the uptake and the efflux of TC were greater in the co-cultures than in the hepatocyte monocultures. Immuno-labelling of sodium-dependent taurocholate transporter (Ntcp) revealed increased expression of the transporter in the presence of KCs. DiscussionHere we presented that KCs have a direct impact on some hepatocyte functions suggesting that the co-culture model may be more suitable for drug related hepatotoxicity studies than hepatocyte monocultures.

Effect of Concentrated Fibroblast-Conditioned Media on In Vitro Maintenance of Rat Primary Hepatocyte.

The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml) showed significantly improved survival and functions (albumin and urea) compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxin- and model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses.

Acetaminophen cytotoxicity is ameliorated in a human liver organotypic co-culture model.

Organotypic liver culture models for hepatotoxicity studies that mimic in vivo hepatic functionality could help facilitate improved strategies for early safety risk assessment during drug development. Interspecies differences in drug sensitivity and mechanistic profiles, low predictive capacity, and limitations of conventional monocultures of human hepatocytes, with high attrition rates remain major challenges. Herein, we show stable, cell-type specific phenotype/cellular polarity with differentiated functionality in human hepatocyte-like C3A cells (enhanced CYP3A4 activity/albumin synthesis) when in co-culture with human vascular endothelial cells (HUVECs), thus demonstrating biocompatibility and relevance for evaluating drug metabolism and toxicity. In agreement with in vivo studies, acetaminophen (APAP) toxicity was most profound in HUVEC mono-cultures; whilst in C3A:HUVEC co-culture, cells were less susceptible to the toxic effects of APAP, including parameters of oxidative stress and ATP depletion, altered redox homeostasis, and impaired respiration. This resistance to APAP is also observed in a primary human hepatocyte (PHH) based co-culture model, suggesting bidirectional communication/stabilization between different cell types. This simple and easy-to-implement human co-culture model may represent a sustainable and physiologically-relevant alternative cell system to PHHs, complementary to animal testing, for initial hepatotoxicity screening or mechanistic studies of candidate compounds differentially targeting hepatocytes and endothelial cells.

Establishment of a hepatocyte-kupffer cell coculture model for assessment of proinflammatory cytokine effects on metabolizing enzymes and drug transporters.

Elevated levels of proinflammatory cytokines associated with infection and inflammation can modulate cytochrome P450 enzymes, leading to potential disease-drug interactions and altered small-molecule drug disposition. We established a human-derived hepatocyte-Kupffer cell (Hep:KC) coculture model to assess the indirect cytokine impact on hepatocytes through stimulation of KC-mediated cytokine release and compared this model with hepatocytes alone. Characterization of Hep:KC cocultures showed an inflammation response after treatment with lipopolysaccharide and interleukin (IL)-6 (indicated by secretion of various cytokines). Additionally, IL-6 exposure upregulated acute-phase proteins (C-reactive protein, alpha-1-acid glycoprotein, and serum amyloid A2) and downregulated CYP3A4. Compared with hepatocytes alone, Hep:KC cocultures showed enhanced IL-1β-mediated effects but less impact from both IL-2 and IL-23. Hep:KC cocultures treated with IL-1β exhibited a higher release of proinflammatory cytokines, an increased upregulation of acute-phase proteins, and a larger extent of metabolic enzyme and transporter suppression. IC50 values for IL-1β-mediated CYP3A4 suppression were lower in Hep:KC cocultures (98.0-144 pg/ml) compared with hepatocytes alone (IC50 > 5000 pg/ml). Cytochrome suppression was preventable by blocking IL-1β interaction with IL-1R1 using an antagonist cytokine or an anti-IL-1β antibody. Unlike IL-1β, IL-6-mediated effects were comparable between hepatocyte monocultures and Hep:KC cocultures. IL-2 and IL-23 caused a negligible inflammation response and a minimal inhibition of CYP3A4. In both hepatocyte monocultures and Hep:KC cocultures, IL-2RB and IL-23R were undetectable, whereas IL-6R and IL-1R1 levels were higher in Hep:KC cocultures. In summary, compared with hepatocyte monocultures, the Hep:KC coculture system is a more robust in vitro model for studying the impact of proinflammatory cytokines on metabolic enzymes.