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

Abstract

BackgroundThe 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% O2: 5% CO2) for up to 6 h. Phosphorescence O2 analyzer was used to determine the rate of cellular mitochondrial O2 consumption (kc, μM O2 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.FindingsRespiration was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. The values of kc (mean ± SD) for 0≤ t ≤6 h were 0.15 ± 0.02 μM O2 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.ConclusionsThe described liver tissue preparation demonstrates stable hepatocyte structure, ultrastructure and biomarkers for up to 6 h, permitting in vitro studies.