Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation:Effects of culture medium

Precision-cut liver slices (PCLS) are an ex vivo model for metabolism and toxicity studies. However, data on the maintenance of the morphological integrity of the various cell types in the slices during prolonged incubation are lacking. Therefore, our aims were to characterize morphological and functional changes in rat PCLS during five days of incubation in a rich medium, RegeneMed®, and a standard medium, Williams' Medium E. Although cells of all types in the slices remain viable, profound changes in morphology were observed, which were more prominent in RegeneMed®. Slices underwent notable fibrosis, bile duct proliferation and fat deposition. Slice thickness increased, resulting in necrotic areas, while slice diameter decreased, possibly indicating cell migration. An increased proliferation of parenchymal and non-parenchymal cells (NPCs) was observed. Glycogen, albumin and Cyp3a1 were maintained albeit to a different level in two media. In conclusion, both hepatocytes and NPCs remain viable and functional, enabling five-day toxicity studies. Tissue remodeling and formation of a new capsule-like cell lining around the slices are evident after 3–4 days. The differences in effects between media emphasize the importance of media selection and of the recognition of morphological changes in PCLS, when interpreting results from toxicological or pharmacological studies

Maintenance of drug metabolism and transport functions in human precision-cut liver slices during prolonged incubation for 5 days

Human precision-cut liver slices (hPCLS) are a valuable ex vivo model that can be used in acute toxicity studies. However, a rapid decline in metabolic enzyme activity limits their use in studies that require a prolonged xenobiotic exposure. The aim of the study was to extend the viability and function of hPCLS to 5 days of incubation. hPCLS were incubated in two media developed for long-term culture of hepatocytes, RegeneMed(®), and Cellartis(®), and in the standard medium WME. Maintenance of phase I and II metabolism was studied both on gene expression as well as functional level using a mixture of CYP isoform-specific substrates. Albumin synthesis, morphological integrity, and glycogen storage was assessed, and gene expression was studied by transcriptomic analysis using microarrays with a focus on genes involved in drug metabolism, transport and toxicity. The data show that hPCLS retain their viability and functionality during 5 days of incubation in Cellartis(®) medium. Albumin synthesis as well as the activity and gene expression of phase I and II metabolic enzymes did not decline during 120-h incubation in Cellartis(®) medium, with CYP2C9 activity as the only exception. Glycogen storage and morphological integrity were maintained. Moreover, gene expression changes in hPCLS during incubation were limited and mostly related to cytoskeleton remodeling, fibrosis, and moderate oxidative stress. The expression of genes involved in drug transport, which is an important factor in determining the intracellular xenobiotic exposure, was also unchanged. Therefore, we conclude that hPCLS cultured in Cellartis(®) medium are a valuable human ex vivo model for toxicological and pharmacological studies that require prolonged xenobiotic exposure

Comparison of Biocompatibility and Adsorption Properties of Different Plastics for Advanced Microfluidic Cell and Tissue Culture Models

Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly­(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision-cut liver slices, that are not possible with conventional systems. However, PDMS, a silicone rubber material, is very hydrophobic and tends to exhibit significant adsorption and absorption of hydrophobic drugs and their metabolites. Although glass could be used as an alternative, thermoplastics are better from a cost and fabrication perspective. Thermoplastic polymers (plastics) allow easy surface treatment and are generally transparent and biocompatible. This study focuses on the fabrication of biocompatible microfluidic devices with low adsorption properties from the thermoplastics poly­(methyl methacrylate) (PMMA), polystyrene (PS), polycarbonate (PC), and cyclic olefin copolymer (COC) as alternatives for PDMS devices. Thermoplastic surfaces were oxidized using UV-generated ozone or oxygen plasma to reduce adsorption of hydrophobic compounds. Surface hydrophilicity was assessed over 4 weeks by measuring the contact angle of water on the surface. The adsorption of 7-ethoxycoumarin, testosterone, and their metabolites was also determined after UV-ozone treatment. Biocompatibility was assessed by culturing human hepatoma (HepG2) cells on treated surfaces. Comparison of the adsorption properties and biocompatibility of devices in different plastics revealed that only UV-ozone-treated PC and COC devices satisfied both criteria. This paper lays an important foundation that will help researchers make informed decisions with respect to the materials they select for microfluidic cell-based culture experiments

Regional Differences in Intestinal Drug Metabolism

The intestines are key for the absorption of nutrients and water as well as drug metabolism, and it is well known that there are clear differences in the expression profile of drug metabolism enzymes along the intestinal tract. Yet, only a few studies have thoroughly investigated regional differences in human intestinal drug metabolism. In this study, we evaluated phase I and phase II metabolism in matched human ileum and colon precision-cut intestinal slices (PCIS). To this end, human PCIS were incubated for 3 h with testosterone (TT) and 7-hydroxycoumarin (7-HC) to examine phase I and phase II metabolism, respectively. Metabolite formation was assessed by high-performance liquid chromatography (HPLC) analysis. Our results demonstrated that androstenedione, 6β-hydroxytestosterone, 2β-hydroxytestosterone, and 7-HC sulfate, were predominantly formed in the ileum, while 15α-hydroxytestosterone and 7-HC glucuronide were mainly produced in the colon. Moreover, we also observed sex differences in phase II metabolite formation, which appeared to be higher in males as compared to females. Taken together, we demonstrated that phase I metabolism predominantly occurs in ileum PCIS, while phase II metabolism mostly takes place in colon PCIS. Moreover, we revealed that human PCIS can be used to study both regional and sex differences in intestinal metabolism

Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu

Hepatic stellate cell (HSC) activation is a key event in the natural process of wound healing as well as in fibrosis development in liver. Current in vitro models for HSC activation contribute significantly to the understanding of HSC biology and fibrogenesis but still fall far short of recapitulating in vivo intercellular functional and anatomic relationships. In addition, when cultured on uncoated plastic, HSC spontaneously activate, which makes HSC activation difficult to regulate or analyze. We have examined whether the use of precision-cut liver slices might overcome these limitations. Liver slices (8 mm diameter, 250 microm thickness) were generated from normal rat liver and incubated for 3 or 16 h with increasing doses of carbon tetrachloride (CCl4). Rat liver slices remained viable during incubation, as shown by minimal enzyme leakage. Expression of markers for HSC activation and the onset of fibrogenesis in the liver slices was studied using real-time PCR and Western blotting. In unstimulated liver slices, mRNA and protein levels of desmin, heat shock protein 47, and alpha B-crystallin remained constant, indicating quiescence of HSC, whereas Krüppel-like factor 6 expression was increased. In contrast, incubation with CCl4 led to a time- and dose-dependent increase in mRNA expression of all markers and an increased alpha B-crystallin protein expression. In conclusion, we have developed a technique to induce activation of quiescent HSC in rat liver slices. This model permits the study of toxicity-induced HSC activation within a physiological milieu, not only in animal but ultimately also in human tissue, and could contribute to the reduction of animal experiment