Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-017-2002-1) contains supplementary material, which is available to authorized users.

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IntroductionOxygen is a critical parameter proposed to modulate the functions of chondrocytes ex-vivo as well as in damaged joints. This article investigates the effect of low (more physiological) oxygen percentage on the biosynthetic and catabolic activity of human articular chondrocytes (HAC) at different phases of in vitro culture.MethodsHAC expanded in monolayer were cultured in pellets for two weeks (Phase I) or up to an additional two weeks (Phase II). In each Phase, cells were exposed to 19% or 5% oxygen. Resulting tissues and culture media were assessed to determine amounts of produced/released proteoglycans and collagens, metalloproteinases (MMPs), collagen degradation products and collagen fibril organization using biochemical, (immuno)-histochemical, gene expression and scanning electron microscopy analyses. In specific experiments, the hypoxia-inducible factor-1α (HIF-1α) inhibitor cadmium chloride was supplemented in the culture medium to assess the involvement of this pathway.ResultsIndependent from the oxygen percentage during expansion, HAC cultured at 5% O2 (vs 19% O2) during Phase I accumulated higher amounts of glycosaminoglycans and type II collagen and expressed reduced levels of MMP-1 and MMP-13 mRNA and protein. Switching to 19% oxygen during Phase II resulted in reduced synthesis of proteoglycan and collagen, increased release of MMPs, accumulation of type II collagen fragments and higher branching of collagen fibrils. In contrast, reducing O2 during Phase II resulted in increased proteoglycan and type II collagen synthesis and reduced expression and release of MMP-13 mRNA and protein. Supplementation of cadmium chloride during differentiation culture at 5% O2 drastically reduced the up-regulation of type II collagen and the down-regulation of MMP-1 mRNA.ConclusionsThe application of more physiologic oxygen percentage during specific phases of differentiation culture enhanced the biosynthetic activity and reduced the activity of catabolic enzymes implicated in cartilage breakdown. Modulation of the oxygen percentage during HAC culture may be used to study pathophysiological events occurring in osteoarthritis and to enhance properties of in vitro engineered cartilaginous tissues.

BackgroundRadiation resistance presents a challenge to the effective treatment of cancer. If therapeutic compounds were capable of resensitizing resistant tumours then a concurrent chemo-radiation treatment could be used to overcome radiation resistance.MethodsWe have developed a phenotypic assay to investigate the response of radiation resistant breast cancer cells grown in 3D-microtissue spheroids to combinations of radiation and established chemotherapeutic drugs. The effects were quantified by real time high content imaging of GFP detection area over 14 days. Ten established chemotherapeutic drugs were tested for their ability to enhance the effects of radiation.ResultsOf ten analysed chemotherapeutics, vinblastine was the most effective compound, with docetaxel and doxorubicine being less effective in combination with radiation. To investigate the response in a model closer to the in vivo situation we investigated the response of heterotypic 3D microtissues containing both fibroblasts and breast cancer cells. Drug treatment of these heterotypic 3D cultures confirmed treatment with radiation plus vinblastine to be additive in causing breast cancer growth inhibition. We have validated the screen by comparing radiation sensitizing effects of known chemotherapeutic agents. In both monotypic and heterotypic models the concurrent treatment of vinblastine and radiation proved more effective inhibitors of mammary cancer cell growth. The effective concentration range of both vinblastine and radiation are within the range used in treatment, suggesting the 3D model will offer a highly relevant screen for novel compounds.ConclusionsFor the first time comfortable 3D cell-based phenotypic assay is available, that allows high throughput screening of compounds with radiation therapy modulating capacity, opening the field to drug discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1481-9) contains supplementary material, which is available to authorized users.

This study aimed at testing whether an RGD-restricted substrate interface is sufficient for adhesion and growth of human articular chondrocytes (HAC), and whether it enhances their post expansion chondrogenic capacity. HAC/ substrate interaction was restricted to RGD by modifying tissue culture polystyrene (TCPS) with a poly(ethylene glycol) (PEG) based copolymer system that renders the surface resistant to protein adsorption while at the same time presenting the bioactive RGD-containing peptide GCRGYGRGDSPG (RGD). As compared to TCPS, HAC cultured on RGD spread faster (1.9-fold), maintained higher type II collagen mRNA expression (4.9-fold) and displayed a 19% lower spreading area. On RGD, HAC attachment efficiency (66±10%) and proliferation rate (0.56±0.04 doublings/day), as well as type II collagen mRNA expression in the subsequent chondrogenic differentiation phase, were similar to those of cells cultured on TCPS. In contrast, cartilaginous matrix deposition by HAC expanded on RGD was slightly but consistently higher (15% higher glycosaminoglycan-to-DNA ratio). RDG (bioinactive peptide) and PEG (no peptide ligand) controls yielded drastically reduced attachment efficiency (lower than 11%) and proliferation (lower than 0.20 doublings/day). Collectively, these data indicate that restriction of HAC interaction with a substrate through RGD peptides is sufficient to support their adhesion, growth and maintenance of cartilage forming capacity. The concept could thus be implemented in materials for cartilage repair, whereby in situ recruited/infiltrated chondroprogenitor cells would proliferate while maintaining their ability to differentiate and generate cartilage tissue.