Optimizing Preincubation Conditions for Precision-cut Rat Kidney and Liver Tissue Slices: Eect of Culture Media and Antioxidants D. K. OBATOMI 1, *, S. BRANT 1 , V. ANTHONYPILLAI 1 , D. A. EARLY 1 and P. H. BACH 2 1 Department of Life Sciences, Faculty of Science and Health, University of East London, Romford Road, London E15 4LZ and 2 BioTechnologic Ltd, Khaya Lami House, Castle Road, Horsell, Woking, Surrey GU21 4EU, UK (Accepted 16 April 1998) AbstractÐTissue slices are commonly `preincubated' before use, but optimal conditions to ensure their maximal viability have not been systematically investigated. The eects of serum-free Dulbecco's mini- mum Eagle's medium and Ham's nutrient mixture (DMEM/F12) (1:1) culture media with and without phenol red ( 2 PR), or RPMI-1640 and six dierent antioxidants on the viability of precision-cut rat kid- ney and liver slices (200 25 mm) 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 main- tained at 378C. In all cases, preincubation produced a time-dependent signi®cant reduction of ethidium bromide positive nuclei stained in each medium and in both kidney and liver slices. Based on lactate de- hydrogenase (LDH) leakage, there are viability dierences between the media. In contrast, alkaline phosphatase (ALP) leakage and MTT reduction were less sensitive and did not dierentiate between slice viability in each incubation medium. Preincubation of kidney and liver slices in DMEM/F12 med- ium containing antioxidants, indicated an enhanced viability which was speci®c for each tissue. Exten- sion 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 ( 2 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 med- ium for maintaining the viability of either kidney or liver slices. However, kidney or liver slices preincu- bated 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 mM in the dierent media for 24 hours showed a signi®cant increase in enzyme leakage, decline of MTT reduc- tive capacity and increased oxidative damage, with toxicity more elaborate in RPMI-1640 medium. Pre- incubation of kidney slices with either reduced glutathione (GSH) or a-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 pro®le. The antioxidants provided partial protection of slices from ATR tox- icity. 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. # 1998 Elsevier Science Ltd. All rights reserved Keywords: improved preincubation conditions; culture media; antioxidants; superoxide dismutase; rat tissue slices; atractyloside; cytotoxicity. Abbreviations: ALP = alkaline phosphatase; ASC=L-ascorbate; ATR = atractyloside; DEF = deferox- amine; DMEM/F12 ( 2 PR) = Dulbecco's minimium Eagle's medium and Ham's nutrient mixture F-12 with or without phenol red; GSH = reduced glutathione; LDH = lactate dehydrogenase; MDA = malondialdehyde; MESNA = 2-mercaptoethanesulfonic acid; PBS = phosphate buered saline; PR = phenol red; SOD = superoxide dismutase; TBA = thiobarbituric acid; TOC = a-tocopherol; TBARS = thiobarbituric acid reactive substance; TCA = trichloroacetic acid. INTRODUCTION Precision-cut tissue slice technology oers a most attractive in vitro system that is already widely used for pharmacotoxicology, metabolism and cell Toxicology in Vitro 12 (1998) 725±737 0887-2333/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(98)00055-1 *Author for correspondence at: Department of Biochemistry, Faculty of Medical Sciences, University of Jos, P.M.B. 2084, Jos, Nigeria.