A preparation of murine liver fragments for in vitro studies: liver preparation for toxicological studies

BACKGROUND: The aim of this study was to develop liver tissue preparation suitable for investigating toxins. Hepatocyte respiration, ATP content, urea synthesis, caspase activity and morphology were measured as a function of in vitro incubation time. Mice were anesthetized by sevoflurane inhalation. Small liver fragments were then rapidly excised and incubated at 37°C in Krebs-Henseleit buffer (continuously gassed with 95% O(2): 5% CO(2)) for up to 6 h. Phosphorescence O(2) analyzer was used to determine the rate of cellular mitochondrial O(2) consumption (k(c), μM O(2) min(-1) mg(-1)). Cellular ATP was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. FINDINGS: Respiration was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. The values of k(c) (mean ± SD) for 0≤ t ≤6 h were 0.15 ± 0.02 μM O(2) min(-1) mg(-1) (n = 18, coefficient of variation, CV = 13%), ATP content 131 ± 69 pmol mg(-1) (1≤ t ≤6 h, n = 16, CV = 53%), synthesized urea 0.134 ± 0.017 mg/dL mg(-1) in 50 min (0≤ t ≤6 h, n = 14, CV = 13%), and AMC peak area 62,540 ± 26,227 arbitrary units mg(-1) (1≤ t ≤6 h, n = 3, CV = 42%). Hepatocyte morphology and organelles were reasonably persevered. CONCLUSIONS: The described liver tissue preparation demonstrates stable hepatocyte structure, ultrastructure and biomarkers for up to 6 h, permitting in vitro studies.