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

Abstract

Doc number: 70

Abstract

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% O₂ : 5% CO₂ ) for up to 6 h. Phosphorescence O₂ analyzer was used to determine the rate of cellular mitochondrial O₂ consumption (k _c , 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.

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 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.

Conclusions: The described liver tissue preparation demonstrates stable hepatocyte structure, ultrastructure and biomarkers for up to 6 h, permitting in vitro studies.