A Unidirectional 96-Well Fluidic Culture Platform for Upstream Cell Dosing with Subsequent Downstream Nonlinear and Ascending Exposure Gradients for Real-Time and Cell-Based Toxicity Screening Environments.

Because of the importance to create screening tools that better mimic models, for exposure responses to drugs or toxicants, reproducible and adaptable culture platforms must evolve as approaches to replicate functions that are native to human organ systems. The Stairstep Waterfall (SsWaterfall) Fluidic Culture System is a unidirectional, multiwell, gravity-driven, cell culture system with micro-channels connecting 12 wells in each row (8-row replicates). The construct allows for the one-way flow of medium, parent and metabolite compounds, and the cellular signaling between connected culture wells while simultaneously operating as a cascading flow and discretized nonlinear dosing device.

Benchmark Concentrations for Untargeted Metabolomics Versus Transcriptomics for Liver Injury Compounds in In Vitro Liver Models.

Interpretation of untargeted metabolomics data from both in vivo and physiologically relevant in vitro model systems continues to be a significant challenge for toxicology research. Potency-based modeling of toxicological responses has served as a pillar of interpretive context and translation of testing data. In this study, we leverage the resolving power of concentration-response modeling through benchmark concentration (BMC) analysis to interpret untargeted metabolomics data from differentiated cultures of HepaRG cells exposed to a panel of reference compounds and integrate data in a potency-aligned framework with matched transcriptomic data.

Comparison of Normalization Methods for Analysis of TempO-Seq Targeted RNA Sequencing Data.

Analysis of bulk RNA sequencing (RNA-Seq) data is a valuable tool to understand transcription at the genome scale. Targeted sequencing of RNA has emerged as a practical means of assessing the majority of the transcriptomic space with less reliance on large resources for consumables and bioinformatics. TempO-Seq is a templated, multiplexed RNA-Seq platform that interrogates a panel of sentinel genes representative of genome-wide transcription.

High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability.

Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult.

Organotypic 3D HepaRG Liver Model for Assessment of Drug-Induced Cholestasis.

Cholestasis remains a major challenge in drug-induced liver injury, and therefore warrants identification of chemical entities that may lead to cholestasis. Recent advances in cell culture methods enable 3D spheroid models to remain viable for much longer periods of time than conventional sandwich cultures of primary human hepatocytes while maintaining native tissue-like functionality, such as drug metabolism activity, receptor signaling functionality, and physiological relevance. These spheroid models enable us to study repeated exposure effects associated with chemicals and their metabolites that may ultimately progress to cholestasis and liver injury.

The Power of Resolution: Contextualized Understanding of Biological Responses to Liver Injury Chemicals Using High-throughput Transcriptomics and Benchmark Concentration Modeling.

Prediction of human response to chemical exposures is a major challenge in both pharmaceutical and toxicological research. Transcriptomics has been a powerful tool to explore chemical-biological interactions, however, limited throughput, high-costs, and complexity of transcriptomic interpretations have yielded numerous studies lacking sufficient experimental context for predictive application. To address these challenges, we have utilized a novel high-throughput transcriptomics (HTT) platform, TempO-Seq, to apply the interpretive power of concentration-response modeling with exposures to 24 reference compounds in both differentiated and non-differentiated human HepaRG cell cultures.

From the Cover: Three-Dimensional (3D) HepaRG Spheroid Model With Physiologically Relevant Xenobiotic Metabolism Competence and Hepatocyte Functionality for Liver Toxicity Screening.

Effective prediction of human responses to chemical and drug exposure is of critical importance in environmental toxicology research and drug development. While significant progress has been made to address this challenge using invitro liver models, these approaches often fail due to inadequate tissue model functionality. Herein, we describe the development, optimization, and characterization of a novel three-dimensional (3D) spheroid model using differentiated HepaRG cells that achieve and maintain physiologically relevant levels of xenobiotic metabolism (CYP1A2, CYP2B6, and CYP3A4/5).

A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies.

Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate losing their hepatocyte functions and metabolic competence. We have developed a three-dimensional in vitro model using extracellular matrix-based hydrogel for long-term culture of the human hepatoma cell line HepG2.

Functional analysis of the porcine USP18 and its role during porcine arterivirus replication.

Emerging evidence places deubiquitylation at the core of a multitude of regulatory processes, ranging from cell growth to innate immune response and health, such as cancer, degenerative and infectious diseases. Little is known about deubiquitylation in pig and arterivirus infection. This report provides information on the biochemical and functional role of the porcine USP18 during innate immune response to the porcine respiratory and reproductive syndrome virus (PRRSV).