Z Gastroenterol 2014; 52 - P_3_06
DOI: 10.1055/s-0033-1360925

Comparison of the response of primary rat and human hepatocytes to ethanol exposition in a miniaturized three-dimensional (3D) multi-compartment bioreactor system

EC Wönne 1, T Hiller 1, J Przibilla 2, U Müller-Vieira 2, R Guthke 3, W Schmidt-Heck 3, G Damm 4, F Knöspel 1, M Richter 1, D koczan 5, AK Nüssler 6, A Schulz 7, K Zeilinger 1
  • 1Charité Universitätsmedizin Berlin, Bioreactor Group, Experimental Surgery, BCRT, Berlin, Germany
  • 2Pharmacelsus GmbH, Saarbrücken, Germany
  • 3Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie, Hans-Knöll-Institut e.V., Jena, Germany
  • 4Charité Universitätsmedizin Berlin, Department of General, Visceral and Transplantation Surgery, Berlin, Germany
  • 5Institute for Immunology/Steinbeis Transfercenter for Proteome Analysis, Core Facility for Microarray Analysis, BMFZ, Rostock, Germany
  • 6Eberhard Karls University Tübingen, Department of Traumatology and Reconstructive Surgery, BG-Trauma and Medical Centre Tübingen, Tübingen, Germany
  • 7Vivantes Humboldt-Klinikum Chirurgie, Visceral- und Gefäßchirurgie, Berlin, Germany

Objective:

Various studies indicate that the accumulation of reactive oxygen species (ROS) and associated cellular damage play an important role in ageing of various organs, including the liver. Especially high concentrations of ethanol lead to oxidative damage, which have been proposed as the major cause of cellular ageing and death. To analyze these effects a bioreactor technology for in vitro liver cell culture was used, which is based on a perfused hollow-fibre capillary network offering dynamic three-dimensional culture conditions with continuous medium perfusion and decentralized oxygenation. This technology creates an in vitro situation, which is close to the physiological conditions in vivo.

The objective of this study was to compare the response to ethanol exposition of primary rat or human hepatocytes cultured in the bioreactor system.

Material and methods:

A number of 30 millions of primary rat hepatocytes from 6 – 8 months or 23 – 25 months old animals, or 20 millions of primary human hepatocytes, respectively, were inoculated in each of four parallel bioreactors under controlled perfusion conditions. At day 3 of cultivation two bioreactors were incubated for 24h or for 48h with medium containing 150 mM, 300 mM or 600 mM ethanol, while control cultures were kept untreated. Liver enzymes and metabolic parameters were measured daily in culture perfusates. To monitor effects of ethanol on gene expression level, Affymetrix Human Gene 1.0 ST Genechips were used for hybridisation.

Results and conclusion:

Prior to ethanol incubation hepatocyte cultures from 6 – 8 months old rats showed a higher initial release of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and glutamate dehydrogenase (GLDH) in comparison to hepatocytes cultures of 23 – 25 months old rats. Regarding metabolic parameters such as glucose, lactate and urea production rate no significant differences between both groups were observed. The incubation of 150 mM ethanol had no significant effect on metabolic or histological parameters in rat or human cultures, while increased ethanol concentrations up to 600 mM for 24h resulted in a decreasing glucose, lactate and urea production rate in human hepatocytes. Focussing on the mTOR- Pathway, the analysis of mRNA revealed distinct effects on gene expression in human cultures, while only minor changes were detected in rat cultures.

The result indicates that the release of typical liver enzymes is dependent on the age in rat hepatocytes. Furthermore human hepatocytes are more sensitive to ethanol exposition than rat hepatocytes and that the effect of ethanol is dose-dependent. Further studies are required to investigate the impact of ethanol exposition on ageing processes in detail.