Fundamentel research and development of 3-dimensional culture models of hepatocytes as an in vitro model for biotransformation and toxiticity studies.

The development of an in vitro model, metabolically functional for a long period of time, is an essential progress in in vitro pharmaco-toxicology. Previous research has focussed on the factors cell-cell interactions and culture media. In this project, a third factor, namely the extracellular matrix, will be evaluated in depth. Collagen gel sandwich configurations and collagen immobilisation gel cultures of hepatocytes are developed as three-dimensional culture models. After resolving basic problems, the expression and regulation of key components of xenobiotic phase 1 and phase 2 biotransformation are investigated. This is realised by measurements of the activity, protein and mRNA levels by enzyme assays with isoenzyme specific substrates, western blots and HPLC protein analyses, northern blots and RNase protection assays, respectively. The necessar methodology will be developed. For phase 1 the mixed function oxidase system and the flavin-containing monooxygenase system are studied; for phase 2 glutathione S-transferase and its isoenzymes. Hormonal regulation, zonation of biotransformation enzymes and the effect of hormones on this zonation are also an important part of this project.