Best Practices and Progress in Precision-Cut Liver Slice Cultures

Thirty-five years ago, precision-cut liver slices (PCLS) were described as a promising tool and were expected to become the standard in vitro model to study liver disease as they tick off all characteristics of a good in vitro model. In contrast to most in vitro models, PCLS retain the complex 3D liver structures found in vivo, including cell–cell and cell–matrix interactions, and therefore should constitute the most reliable tool to model and to investigate pathways underlying chronic liver disease in vitro. Nevertheless, the biggest disadvantage of the model is the initiation of a procedure-induced fibrotic response. In this review, we describe the parameters and potential of PCLS cultures and discuss whether the initially described limitations and pitfalls have been overcome. We summarize the latest advances in PCLS research and critically evaluate PCLS use and progress since its invention in 1985.

Chitosan-Glutathione Nanoparticles Modify Hepatic Cellular Response on Bovine Precision-cut Liver Slices Treated With Zilpaterol and Clenbuterol.

Zilpaterol and clenbuterol are two β-adrenergic agonist drugs used on 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 at 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 proven yet of zilpaterol to produce reactive oxygen (ROS) and nitrogen species. Regarding glutathione which is the main intracellular antioxidant and plays detoxification functions on liver metabolism; case study is a primary 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 exposed to clenbuterol and zilpaterol. A single drug assay was performed in the 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 agonist β-adrenergic modify in a dose-dependent manner oxide-reduction response through ROS generation, glutathione peroxidase activity, and intracellular GSH content; and de release of liver enzymes associated with hepatocellular damage like gamma glutamyl-transpeptidase, aspartate aminotransferase, alanine aminotransferase. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

Investigating oxygen transport efficiencies in precision-cut liver slice-based organ-on-a-chip devices

Microfluidic ‘organ-on-a-chip’ devices hold great potential for better mimicking the continuous flow microenvironment experienced by tissue and cells in vivo, thereby ensuring realistic transport of nutrients and elimination of waste products. However, the mass transport of oxygen, which arguably is the most critical nutrient due to its inherently low solubility in water, is rarely assessed. To this aim, the suitability of various precision-cut liver slice (PCLS) microfluidic devices for the defined maintenance of oxygen mass transport were evaluated using COMSOL simulations, leading to the development of a novel, optimised design to provide defined in vivo oxygenation conditions within an organ-on-a-chip system. Simulations found that the proposed device was capable of maintaining 43% of the tissue slice volume within the physiological range of the liver against 18% for the best performing literature device. The optimal device architecture derived from the modelling was then fabricated and its operation confirmed with an LDH assay. These simulation results form the basis for a greater understanding of not just the challenges involved in designing organ-on-a-chip devices, but also highlight issues that would arise from the incorporation of additional organs, as research progresses towards complete human-on-a-chip model systems.

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.

Real-World Experience Measurement of Liver Iron Concentration by R2 vs. R2 Star MRI in Hemoglobinopathies

Background: Non-invasive determination of liver iron concentration (LIC) is a valuable tool that guides iron chelation therapy in transfusion-dependent patients. Multiple methods have been utilized to measure LIC by MRI. The purpose of this study was to compare free breathing R2* (1/T2*) to whole-liver Ferriscan R2 method for estimation of LIC in a pediatric and young adult population who predominantly have hemoglobinopathies. Methods: Clinical liver and cardiac MRI scans from April 2016 to May 2018 on a Phillips 1.5 T scanner were reviewed. Free breathing T2 and T2* weighted images were acquired on each patient. For T2, multi-slice spin echo sequences were obtained. For T2*, a single mid-liver slice fast gradient echo was performed starting at 0.6 ms with 1.2 ms increments with signal averaging. R2 measurements were performed by Ferriscan analysis. R2* measurements were performed by quantitative T2* map analysis. Results: 107 patients underwent liver scans with the following diagnoses: 76 sickle cell anemia, 20 Thalassemia, 9 malignancies and 2 Blackfan Diamond anemia. Mean age was 12.5 ± 4.5 years. Average scan time for R2 sequences was 10 min, while R2* sequence time was 20 s. R2* estimation of LIC correlated closely with R2 with a correlation coefficient of 0.94. Agreement was strongest for LIC < 15 mg Fe/g dry weight. Overall bias from Bland–Altman plot was 0.66 with a standard deviation of 2.8 and 95% limits of agreement −4.8 to 6.1. Conclusion: LIC estimation by R2* correlates well with R2-Ferriscan in the pediatric age group. Due to the very short scan time of R2*, it allows imaging without sedation or anesthesia. Cardiac involvement was uncommon in this cohort.

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.