title: Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium.
abstract: Isolated hepatocytes in suspension express most of the functional activities of the intact liver and offer an easy-to-handle in vitro system for investigating both the biotransformation and damaging effects induced after a single exposure to xenobiotics upto 3-4h. There is, however, a general lack of consensus with respect to the choice of a suitable suspension medium. This motivated us to perform a comparative study of the effects of five frequently used bicarbonate-based media (Ca(2+)-containing Krebs-Henseleit buffer (KHB) with or without 25mM HEPES, 10mM glucose and 2% (g/v) BSA supplements, and Williams' E culture medium) on the viability (LDH leakage, caspase-3 processing and activity, Bid/Bax expression) and functionality (energy status, glutathione content, phases I and II biotransformation) of freshly isolated rat hepatocytes in suspension upto 3h. Also included was the bicarbonate-free HEPES buffer that does not require carbogen gassing, and is therefore handled more easily. The results clearly demonstrated that the type of incubation medium profoundly affected the functionality of the suspended hepatocytes, changing their sensitivity and response to exogenous damaging effects. While HEPES buffer and Williams' E medium offered the lowest background of spontaneous cell death, bicarbonate-based buffers and media seemed more suitable for obtaining both phases I and II biotransformation. Williams' E medium ensured a constant glutathione content of the cells and a lower level of oxidative stress.
authors.affiliation: Department of Toxicology, Pharmaceutical Institute, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. greetje.elaut@vub.ac.be <greetje.elaut@vub.ac.be>
authors.forename: Greetje
authors.initials: G
authors.lastname: Elaut
authors.forename: Tamara
authors.initials: T
authors.lastname: Vanhaecke
authors.forename: Yvan Vander
authors.initials: YV
authors.lastname: Heyden
authors.forename: Vera
authors.initials: V
authors.lastname: Rogiers
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001711:Biotransformation / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology*; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D009336:Necrosis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D003470:Culture Media; D005978:Glutathione