Optimizing preincubation conditions for precision-cut rat kidney and liver tissue slices: effect of culture media and antioxidants

Abstract

Tissue slices are commonly ‘preincubated’ before use, but optimal conditions to ensure their maximal viability have not been systematically investigated. The effects of serum-free Dulbecco's minimum Eagle's medium and Ham's nutrient mixture (DMEM/F12) (1:1) culture media with and without phenol red (±PR), or RPMI-1640 and six different antioxidants on the viability of precision-cut rat kidney and liver slices (200±5 μm) were investigated. Slice viability was assessed every 30 minutes over a 2-hour preincubation period and after 24 hours of incubation in a multiwell plate culture system maintained at 37°C. In all cases, preincubation produced a time-dependent significant reduction of ethidium bromide positive nuclei stained in each medium and in both kidney and liver slices. Based on lactate dehydrogenase (LDH) leakage, there are viability differences between the media. In contrast, alkaline phosphatase (ALP) leakage and MTT reduction were less sensitive and did not differentiate between slice viability in each incubation medium. Preincubation of kidney and liver slices in DMEM/F12 medium containing antioxidants, indicated an enhanced viability which was specific for each tissue. Extension of the culture period to 24 hours after 1 hour of preincubation showed up to a further 4–13% leakage of ALP or LDH in DMEM/F12 (±PR) media for both kidney and liver slices and with a further 5–15% decline in MTT viability assay. RPMI-1640 medium on its own was not a suitable medium for maintaining the viability of either kidney or liver slices. However, kidney or liver slices preincubated with DMEM/F12 medium in the presence of some of the antioxidants were satisfactorily maintained for 24 hours. Exposure of slices to atractyloside (ATR) at concentrations of 0.2–2.0 m m in the different media for 24 hours showed a significant increase in enzyme leakage, decline of MTT reductive capacity and increased oxidative damage, with toxicity more elaborate in RPMI-1640 medium. Preincubation of kidney slices with either reduced glutathione (GSH) or α-tocopherol (TOC) and liver slices with either GSH or deferoxamine (DEF) followed by 24 hours of exposure to ATR showed a similar decline in toxicity profile. The antioxidants provided partial protection of slices from ATR toxicity. The results demonstrate the importance of slice preincubation and indicate that slices could be maintained in culture using an appropriate medium, thus providing slices that could serve as a useful alternative in vitro system for assessing novel compounds for toxicity.