The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status

Sandra Coecke, Vera Rogiers, Martin Bayliss, José Castell, Johannes Doehmer, Gérard Fabre, Jeffrey Fry, Armin Kern and Carl Westmoreland

In this report, metabolically competent in vitro systems have been reviewed, in the context of drug metabolising enzyme induction. Based on the experience of the scientists involved, a thorough survey of the literature on metabolically competent long-term culture models was performed. Following this, a prevalidation proposal for the use of the collagen gel sandwich hepatocyte culture system for drug metabolising enzyme induction was designed, focusing on the induction of the cytochrome P450 enzymes as the principal enzymes of interest. The ultimate goal of this prevalidation proposal is to provide industry and academia with a metabolically competent in vitro alternative for long-term studies. In an initial phase, the prevalidation study will be limited to the investigation of induction. However, proposals for other long-term applications of these systems should be forwarded to the European Centre for the Validation of Alternative Methods for consideration. The prevalidation proposal deals with several issues, including: a) species; b) practical prevalidation methodology; c) enzyme inducers; and d) advantages of working with independent expert laboratories. Since it is preferable to include other alternative tests for drug metabolising enzyme induction, when such tests arise, it is recommended that they meet the same level of development as for the collagen gel sandwich long-term hepatocyte system. Those tests which do so should begin the prevalidation and validation process.
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Three-dimensional Co-culture of Primary Human Liver Cells in Bioreactors for In Vitro Drug Studies: Effects of the Initial Cell Quality on the Long-term Maintenance of Hepatocyte-specific Functions

Katrin Zeilinger, Igor M. Sauer, Gesine Pless, Catrin Strobel, Jeannette Rudzitis, Aiguo Wang, Andres K. Nüssler, Alexander Grebe, Lei Mao, Stefan H.G. Auth, Juliane Unger, Peter Neuhaus and Jörg C. Gerlach

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation. We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells. The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation. In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (P450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture. Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value. In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture. After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver. Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture. Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor. This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.
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The Response of a Co-culture Lung Model to Fine and Ultrafine Particles of Incinerator Fly Ash at the Air–liquid Interface

Silvia Diabaté, Sonja Mülhopt, Hanns-Rudolf Paur and Harald F. Krug

Elevated concentrations of particulate matter in the environmental atmosphere constitute a potential risk to human health. In vitro cell-based assays are therefore necessary to evaluate the toxicological potential of inhaled particulate emissions. In this study, the exposure of a co-culture cell model at the air–liquid interface was used to evaluate the dose-dependent biological effects of a test aerosol. The CULTEX ® system was used to expose human cells to an environmentally-relevant aerosol, generated from fly ash collected in a commercial municipal waste incinerator and resuspended in filtered air. Human bronchial epithelial cells, BEAS-2B, co-cultured with differentiated THP-1 macrophages growing on Transwell® inserts, were employed in the bioassay. Analyses of cell viability, interleukin-8 (IL-8) release, intracellular glutathione, and haeme oxygenase-1 enzyme expression were performed. Transportation of the cells and exposure to humidified filtered air or the test aerosol, at 100ml/min for 1 to 6 hours, were well tolerated by the cells and had no effect on their viability. Levels of IL-8 release and haeme oxygenase-1 expression were elevated by exposure to fly ash aerosol as a function of time, but not by exposure to clean air. For IL-8 release, a dose-dependent effect was demonstrated with the assumption that the deposited mass of the particles correlated with exposure time. Exposure to the test aerosol did not affect the intracellular glutathione concentration. This in vitro approach simulates particle deposition in the human lung more realistically than does submerged exposure, and it preserves the inherent properties of the particles. It shows promise for use to detect particulate emissions which are potentially detrimental to human health.
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