Cultured Liver Slices Research Articles

Optimized and Simplified Technique for the Production and Culture of Precision-Cut Liver Slices.

This protocol presents a simple system for the creation and culture of Precision-cut Liver Slices (PCLS). PCLS contains all cells in an intact environment and, therefore, resembles a mini model of the whole organ. They enable the study of live tissues while replicating their complex phenotypes. This protocol allows the preparation of slices from mouse livers using a vibratome and standard laboratory equipment. Protocols for producing and culturing PCLS lack standardization and can vary quite drastically depending on the tissue of interest, the type of vibratome used, and the need for oxygen. These can be difficult to reproduce in some laboratories that have only access to a basic vibratome and common tissue culture facilities. We have put together a protocol focusing on the importance of some key steps within the varied protocols already available. This protocol, therefore, emphasizes the importance of the embedding method, the cutting orientation, a dynamic versus a static system, and the relevance of a minimum volume of culture. This protocol can be established and reproduced in a simple manner in most laboratories that have access to a basic tissue slicer. Taken together and following this protocol, PCLS can stay alive for a minimum of 4 days. PCLS is a simple, economical, and reproducible model to study pathophysiological and therapeutic screening for organs such as the liver.

Transcriptome responses to benzo[a]pyrene in liver slices of sub-arctic fish species

Due to the expanding oil-related activities, the arctic and sub-arctic marine environments are increasingly vulnerable to oil-related pollution such as accidental oil spills. These cold-water ecosystems harbor many fish species that are both ecologically and economically important such as the pelagic polar cod (Boreogadus saida), capelin (Mallotus villosus), and benthic long rough dab (Hippoglossoides platessoides). The latter two are much less studied and it is crucial to characterize their responses to oil-related contaminants and develop molecular biomarkers and genomic resources for future monitoring. In this study, liver slice preparation and culture methods were used to characterize the transcriptome responses (using RNA-seq) in capelin and long rough dab to exposures of the polycyclic aromatic hydrocarbon (PAH) compound benzo[a]pyrene (BaP). The liver slice culture and exposure experiments were performed onboard a research vessel in the Barents Sea. Strong up-regulation of genes involved in biotransformation, particularly the aryl hydrocarbon receptor signaling pathway was observed in both species. A comparison of the number of differentially expressed genes (DEGs) with previously published polar cod exposures indicates that the latter responded more strongly (higher number of genes), suggesting higher uptake and bioconcentration of BaP in the fatty liver tissue, although other factors such as differences in clearance rate could potentially affect the responses. This study provides new genomic resources and gene expression biomarkers in capelin and long rough dab, enhancing our understanding of their response mechanism to oil-related contaminants.

LX-2 Stellate Cells Are a Model System for Investigating the Regulation of Hepatic Vitamin A Metabolism and Respond to Tumor Necrosis Factor α and Interleukin 1β.

Hepatic stellate cells (HSCs) are the major site of vitamin A (retinol) esterification and subsequent storage as retinyl esters within lipid droplets. However, retinyl esters become depleted in many pathophysiological states, including acute and chronic liver injuries. Recently, using a liver slice culture system as a model of acute liver injury and fibrogenesis, a time-dependent increase and decrease in the apparent formation of the bioactive retinoid all-trans-retinoic acid (atRA) and retinyl palmitate was measured, respectively. This coincided with temporal changes in the gene expression of retinoid-metabolizing enzymes and binding proteins, that preceded HSC activation. However, the underlying mechanisms that promote early changes in retinoid metabolism remain unresolved. We hypothesized that LX-2 cells could be applied to investigate differences in quiescent and activated HSC retinoid metabolism. We demonstrate that the hypermetabolic state of activated stellate cells relative to quiescent stellate cells may be attributed to induction of STRA6, RBP4, and CYP26A1, thereby reducing intracellular concentrations of atRA. We further hypothesized that paracrine and autocrine cytokine signaling regulates HSC vitamin A metabolism in both quiescent and activated cells. In quiescent cells, tumor necrosis factor α dose-dependently downregulated LRAT and CRBP1 mRNA, with EC50 values of 30-50 pg/mL. Likewise, interleukin-1β decreased LRAT and CRBP1 gene expression but with less potency. In activated stellate cells, multiple enzymes were downregulated, suggesting that the full effects of altered hepatic vitamin A metabolism in chronic conditions require both paracrine and autocrine signaling events. Further, this study suggests the potential for cell type-specific autocrine effects in hepatic retinoid signaling. SIGNIFICANCE STATEMENT: HSCs are the major site of vitamin A storage and important determinants of retinol metabolism during liver fibrogenesis. Here, two LX-2 culture methods were applied as models of hepatic retinoid metabolism to demonstrate the effects of activation status and dose-dependent cytokine exposure on the expression of genes involved in retinoid metabolism. This study suggests that compared to quiescent cells, activated HSCs are hypermetabolic and have reduced apparent formation of retinoic acid, which may alter downstream retinoic acid signaling.

Human Precision-Cut Liver Slices: A Potential Platform to Study Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

Abstract 6152: Exploiting ferroptosis in mutant BAP1 uveal melanoma

Abstract Uveal melanoma (UM) arises from melanocytes of the uveal tract in the eye and is the most common intraocular malignancy in adults. Primary UM lesions often harbor mutations in G-proteins, GNAQ or GNA11, and are successfully treated by radiotherapy or enucleation. However, 50% of patients develop metastases. Of these metastatic UM (MUM) patients, 80%develop metastases to the liver, an effect strongly correlated with the loss of function of BAP1,a nuclear deubiquitinase. BAP1-mutant MUMs are less responsive to conventional treatments, such as chemotherapy and immune checkpoint inhibitors, with 50% of these patients having an overall survival of only 1 year. We propose that the unique metabolic intermediates found in liver microenvironment may dictate MUM tropism, aggressiveness, and poor therapeutic response. Here, using a combination of in vitro and ex vivo liver slice cultures, we found that the liver microenvironment induces gene expression alterations consistent with limiting the toxicity of reactive oxygen species (ROS). Importantly, we note that mutant BAP1 UM displayed improved ROS detoxification compared to its wildtype counterparts. Furthermore, publicly available datasets suggest patients with mutant BAP1 UM, the subset of patients with liver metastases, have corresponding ROS detoxification gene expression patterns. These findings suggest that the liver microenvironment and BAP1 loss directs MUM to mitigate ROS-associated toxicity. This compensatory mechanism may be therapeutically exploited and pair well as a combination treatment with the recently approved bispecific agent, Tebentafusp, to selectively target liver MUM. Citation Format: Camille J. Cunanan. Exploiting ferroptosis in mutant BAP1 uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6152.

In Vitro Hepatoprotective Activity of Methanolic Leaf Extract of Acalypha indica Against CCl4 Induced Hepatotoxicity in Goat Liver Slice Culture

Plants used in traditional medicine may constitute an important source of new biologically active compounds. Acalypha indica is one of the weed plants that contain important medicinal values for human health applications. The present study was undertaken to assess the hepatoprotective activity of methanolic leaf extract of Acalypha indica in goat liver slice culture against carbon tetrachloride (CCl4) to prove its efficacy against liver disorders CCl4 was used to induce hepatotoxicity in liver slice of goat. The cytotoxicity induced by CCl4 was estimated by quantifying the release of marker enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), acid phosphatase (ACP) and lactate dehydrogenase (LDH). Also, the degree of hepatic damage was measured by estimating the levels of lipid peroxidation (LPO) of hepatocytes membrane lipids. The treatment of liver cells with CCl4 caused twice increase in LPO of cells besides release of LDH, ALT, AST, ACP and ALP by 6.55, 3.89, 3.31, 2.69 and 2.50 times, respectively as compared to untreated liver cells. Thus, a toxic effect of CCl4 was significantly reduced by the treatment of methanolic leaf extract of Acalypha indica, silymarin and quercetin. The free radical scavenging activity of plant extract was estimated by 2, 2-diphenylpicrylhydrazyl (DPPH) method and IC50 is 65.12 ± 2.27 μg/mL. Qualitative analysis of flavonoid indicates the presence of flavonoids in plant extract. These results indicate that all concentration of plant extract of Acalypha indica, silymarin and quercetin protect the liver cells from CCl4 induced oxidative/free radical mediated damage in vitro. The hepatoprotective activity of methanolic extract of Acalypha indica extract is due to the presence of flavonoids which have remarkable antioxidant and anti-inflammatory activity. HIGHLIGHTS Liver disorders are the most common health hazard found in developing countries due to dietary habits, alcohol ingestion, poor hygiene, unsupervised drug use and smoking. Liver diseases can be non-inflammatory, inflammatory and degenerative Acalypha indica is one of the weed plants that contain important medicinal values for human health applications In vitro study of methanolic leaf extract of Acalypha indica against CCl4 induced hepatic damage in goat liver slice culture proven its efficacy against liver diseases GRAPHICAL ABSTRACT

IL-17A in Human Liver: Significant Source of Inflammation and Trigger of Liver Fibrosis Initiation.

IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFβ1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0–F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A+ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers’ analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A+INFγ+ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.

2D and 3D liver models

2D and 3D liver models

Improved Precision-Cut Liver Slice Cultures for Testing Drug-Induced Liver Fibrosis.

In vitro models of human liver disease often fail to mimic the complex 3D structures and cellular organizations found in vivo. Precision cut liver slices (PCLS) retain the complex physiological architecture of the native liver and therefore could be an exceptional in vitro liver model. However, the production of PCLS induces a spontaneous culture-induced fibrogenic reaction, limiting the application of PCLS to anti-fibrotic compounds. Our aim was to improve PCLS cultures to allow compound-induced fibrosis induction. Hepatotoxicity in PCLS cultures was analyzed by lactate dehydrogenase leakage and albumin secretion, while fibrogenesis was analyzed by qRT-PCR and western blot for hepatic stellate cell (HSC) activation markers and collagen 6 secretion by enzyme-linked immunosorbent assays (ELISA). We demonstrate that supplementation of 3 mm mouse PCLS cultures with valproate strongly reduces fibrosis and improves cell viability in our PCLS cultures for up to 5 days. Fibrogenesis can still be induced both directly and indirectly through exposure to TGFβ and the hepatotoxin acetaminophen, respectively. Finally, human PCLS cultures showed similar but less robust results. In conclusion, we optimized PCLS cultures to allow for drug-induced liver fibrosis modeling.

Response of Human Liver Tissue to Innate Immune Stimuli.

Precision-cut human liver slice cultures (PCLS) have become an important alternative immunological platform in preclinical testing. To further evaluate the capacity of PCLS, we investigated the innate immune response to TLR3 agonist (poly-I:C) and TLR4 agonist (LPS) using normal and diseased liver tissue. Pathological liver tissue was obtained from patients with active chronic HCV infection, and patients with former chronic HCV infection cured by recent Direct-Acting Antiviral (DAA) drug therapy. We found that hepatic innate immunity in response to TLR3 and TLR4 agonists was not suppressed but enhanced in the HCV-infected tissue, compared with the healthy controls. Furthermore, despite recent HCV elimination, DAA-cured liver tissue manifested ongoing abnormalities in liver immunity: sustained abnormal immune gene expression in DAA-cured samples was identified in direct ex vivo measurements and in TLR3 and TLR4 stimulation assays. Genes that were up-regulated in chronic HCV-infected liver tissue were mostly characteristic of the non-parenchymal cell compartment. These results demonstrated the utility of PCLS in studying both liver pathology and innate immunity.

Precision-cut liver slices as a model for assess hepatic cellular response of chitosan–glutathione nanoparticles on cultures treated with zilpaterol and clenbuterol

Zilpaterol and clenbuterol are two β-adrenergic agonist drugs used in animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries. Clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used in unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proved yet of zilpaterol to produce reactive oxygen and nitrogen species. Regarding glutathione which is the main intracellular antioxidant plays detoxification functions on liver metabolism; in this work, it is our interest to know the capacity of chitosan–glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures (PCLS) exposed to clenbuterol and zilpaterol. A single drug assay was performed in first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both β-adrenergic agonists modify in a dose-dependent manner in oxide-reduction response through ROS generation. The activity or content of glutathione peroxidase activity, intracellular GSH, gamma glutamyl-transpeptidase, aspartate aminotrasnferase and alanine aminotrasnferase were modified. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

A conserved pathway of transdifferentiation in murine Kupffer cells.

Abundant long-lived liver-resident macrophages, termed Kupffer cells, are activated during chronic liver injury. They secrete both pro-inflammatory and pro-fibrotic cytokines, which act on hepatic stellate cells causing their transdifferentiation into myofibroblasts that deposit collagen. In other tissues, wound-associated macrophages go further, and transdifferentiate into fibrocytes, secreting collagen themselves. We tested Kupffer cells for this property in two experimental models: mixed non-parenchymal cell culture, and precision-cut liver slice culture. Using the Emr1-Cre transgene as a driver and the RiboTag transgene as a reporter, we found that Kupffer cells undergo transdifferentiation under these circumstances. Over time, they lose the expression of both Kupffer cell-specific and macrophage-specific genes and the transcription factors that control their expression, and they begin to express multiple genes and proteins characteristic of either myofibroblasts or tissue fibroblasts. These effects were strongly conserved between non-parenchymal cell culture and liver tissue slice culture, arguing that such transdifferentiation is a conserved function of Kupffer cells. We conclude that in addition to supporting fibrosis through an action on stellate cells, Kupffer cells also participate in liver fibrosis through transdifferentiation into fibrocytes.

Transcriptome responses in polar cod (Boreogadus saida) liver slice culture exposed to benzo[a]pyrene and ethynylestradiol: insights into anti-estrogenic effects

Polar cod (Boreogadus saida) is a key species in the arctic marine ecosystem vulnerable to effects of pollution, particularly from petroleum related activities. To facilitate studying the effects of those pollutants, we adapted a precision-cut liver slice culture protocol for this species. Using this system on board a research vessel, we studied gene expression in liver slice after exposure to the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP), ethynylestradiol (EE2), and their mixtures, to map their molecular targets and examine possible anti-estrogenic effects of BaP. The exposure experiments were performed with BaP alone (0.1, 1, and 10 μM) or in combination with low concentrations of EE2 (5 nM) to mimic physiological estradiol levels in early vitellogenic female fish. Transcriptome analysis (RNA-seq) was performed after 72 h exposure in culture to map the genes and cellular pathways affected. The results provide a view of global transcriptome responses to BaP and EE2, which resulted in enrichment of many pathways such as the aryl hydrocarbon (Ahr) and estrogen receptor pathways. In the mixture exposure, BaP resulted in anti-estrogenic effects, shown by attenuation of EE2 activated transcription of many estrogen target genes. The results from this ex vivo experiment suggest that pollutants that activate the Ahr pathway such as the PAH compound BaP can result in anti-estrogenic effects that may lead to endocrine disruption in polar cod.

Adult human liver slice cultures: Modelling of liver fibrosis and evaluation of new anti-fibrotic drugs.

BACKGROUNDLiver fibrosis can result in end-stage liver failure and death.AIMTo examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model.METHODSFibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected from patients after liver resection. Human liver slices (HLS) were cultivated for up to 21 days. Hepatitis C virus (HCV) infection, alcohol (ethanol stimulation) and steatosis (palmitate stimulation) were examined in fibrotic (F2 to F4) liver slices infected (or not) with HCV. F0-F1 HLS were used as controls. At day 0, either ursodeoxycholic acid (choleretic and hepatoprotective properties) and/or α-tocopherol (antioxidant properties) were added to standard of care on HLS and fibrotic liver slices, infected (or not) with HCV. Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay.RESULTSThe cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs. Expression of the biomarkers of fibrosis and the progression to steatosis (estimated by triglycerides production) was increased with the addition of HCV and /or ethanol or palmitate. From day 15 of the follow-up studies, a significant decrease of both transforming growth factor β-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.CONCLUSIONThese results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver (viral, alcoholic, nonalcoholic steatohepatitis liver diseases) and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination.

Liver Abnormalities after Elimination of HCV Infection: Persistent Epigenetic and Immunological Perturbations Post-Cure.

Chronic hepatitis C (CHC) is a major cause of hepatocellular carcinoma (HCC) worldwide. While directly acting antiviral (DAA) drugs are now able to cure virtually all hepatitis C virus (HCV) infections, even in subjects with advanced liver disease, what happens to the liver and progression of the disease after DAA-induced cure of viremia is only beginning to emerge. Several large-scale clinical studies in different patient populations have shown that patients with advanced liver disease maintain a risk for developing HCC even when the original instigator, the virus, is eliminated by DAAs. Here we review emerging studies derived from multiple, complementary experimental systems involving patient liver tissues, human liver cell cultures, human liver slice cultures, and animal models, showing that HCV infection induces epigenetic, signaling, and gene expression changes in the liver associated with altered hepatic innate immunity and liver cancer risk. Of critical importance is the fact that these virus-induced abnormalities persist after DAA cure of HCV. These nascent findings portend the discovery of pathways involved in post-HCV immunopathogenesis, which may be clinically actionable targets for more comprehensive care of DAA-cured individuals.

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.

Precision-cut human liver slice cultures as an immunological platform

The liver is the central metabolic organ in the human body, and also plays an essential role in innate and adaptive immunity. While mouse models offer significant insights into immune-inflammatory liver disease, human immunology differs in important respects. It is not easy to address those differences experimentally. Therefore, to improve the understanding of human liver immunobiology and pathology, we have established precision-cut human liver slices to study innate immunity in human tissue. Human liver slices collected from resected livers could be maintained in ex vivo culture over a two-week period. Although an acute inflammatory response accompanied by signs of tissue repair was observed in liver tissue following slicing, the expression of many immune genes stabilized after day 4 and remained stable until day 15. Remarkably, histological evidence of pre-existing liver diseases was preserved in the slices for up to 7 days. Following 7 days of culture, exposure of liver slices to the toll-like receptor (TLR) ligands, TLR3 ligand Poly-I:C and TLR4 ligand LPS, resulted in a robust activation of acute inflammation and cytokine genes. Moreover, Poly-I:C treatment induced a marked antiviral response including increases of interferons IFNB, IL-28B and a group of interferon-stimulated genes. Therefore, precision-cut liver slices emerge as a valuable tool to study human innate immunity.

Gut Microbiota-Derived Short Chain Fatty Acids Induce Circadian Clock Entrainment in Mouse Peripheral Tissue

Microbiota-derived short-chain fatty acids (SCFAs) and organic acids produced by the fermentation of non-digestible fibre can communicate from the microbiome to host tissues and modulate homeostasis in mammals. The microbiome has circadian rhythmicity and helps the host circadian clock function. We investigated the effect of SCFA or fibre-containing diets on circadian clock phase adjustment in mouse peripheral tissues (liver, kidney, and submandibular gland). Initially, caecal SCFA concentrations, particularly acetate and butyrate, induced significant day-night differences at high concentrations during the active period, which were correlated with lower caecal pH. By monitoring luciferase activity correlated with the clock gene Period2 in vivo, we found that oral administration of mixed SCFA (acetate, butyrate, and propionate) and an organic acid (lactate), or single administration of each SCFA or lactate for three days, caused phase changes in the peripheral clocks with stimulation timing dependency. However, this effect was not detected in cultured fibroblasts or cultured liver slices with SCFA applied to the culture medium, suggesting SCFA-induced indirect modulation of circadian clocks in vivo. Finally, cellobiose-containing diets facilitated SCFA production and refeeding-induced peripheral clock entrainment. SCFA oral gavage and prebiotic supplementation can facilitate peripheral clock adjustment, suggesting prebiotics as novel therapeutic candidates for misalignment.

3D in vitro models of liver fibrosis.

Animal testing is still the most popular preclinical assessment model for liver fibrosis. To develop efficient anti-fibrotic therapies, robust and representative in vitro models are urgently needed. The most widely used in vitro fibrosis model is the culture-induced activation of primary rodent hepatic stellate cells. While these cultures have contributed greatly to the current understanding of hepatic stellate cell activation, they seem to be inadequate to cover the complexity of this regenerative response. This review summarizes recent progress towards the development of 3D culture models of liver fibrosis. Thus far, only a few hepatic culture systems have successfully implemented hepatic stellate cells (or other non-parenchymal cells) into hepatocyte cultures. Recent advances in bioprinting, spheroid- and precision-cut liver slice cultures and the use of microfluidic bioreactors will surely lead to valid 3D in vitro models of liver fibrosis in the near future.

Studying human hepatic immunology through the isolation and culture of liver slices and purified cellular subpopulations

Abstract Although advances in culture techniques have allowed us to better understand hepatocyte inflammatory signaling under both normal and diseased conditions, still little is known about how non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and hepatic macrophages (i.e. Kupffer cells) impact these events in the intact human liver. In order to investigate this network of cellular cross-talk, we have developed two novel ex vivo culture systems. In Method A, 250 μm slices of human liver tissue were cultured in a transwell system. These slices induced inflammatory and tissue repair signals following both physical injury and challenge with pro-inflammatory ligands (polyI:C, LPS, and LTA). Responses following physical injury were consistent with an acute phase response, while pro-inflammatory ligands induced responses consistent with TLR activation and production of type I interferon. In Method B, we perfused wedges of liver tissue and isolated purified hepatocyte and NPC populations. Hepatocytes isolated by this procedure were successfully maintained in culture for up to 14 days on collagen-coated plates. Isolated NPCs were further purified by FACS analysis into individual populations of LSECs, HSCs, and Kupffer cells. Purified Kupffer cells recovered from sorting were also successfully maintained in culture. Together, these systems will ultimately allow us to examine how the presence of intact liver structure and/or various NPC populations impacts liver cellular signaling in response to different immune stimuli.