long-term culture

/Tag:long-term culture

The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status

Sandra Coecke, Vera Rogiers, Martin Bayliss, José Castell, Johannes Doehmer, Gérard Fabre, Jeffrey Fry, Armin Kern and Carl Westmoreland

In this report, metabolically competent in vitro systems have been reviewed, in the context of drug metabolising enzyme induction. Based on the experience of the scientists involved, a thorough survey of the literature on metabolically competent long-term culture models was performed. Following this, a prevalidation proposal for the use of the collagen gel sandwich hepatocyte culture system for drug metabolising enzyme induction was designed, focusing on the induction of the cytochrome P450 enzymes as the principal enzymes of interest. The ultimate goal of this prevalidation proposal is to provide industry and academia with a metabolically competent in vitro alternative for long-term studies. In an initial phase, the prevalidation study will be limited to the investigation of induction. However, proposals for other long-term applications of these systems should be forwarded to the European Centre for the Validation of Alternative Methods for consideration. The prevalidation proposal deals with several issues, including: a) species; b) practical prevalidation methodology; c) enzyme inducers; and d) advantages of working with independent expert laboratories. Since it is preferable to include other alternative tests for drug metabolising enzyme induction, when such tests arise, it is recommended that they meet the same level of development as for the collagen gel sandwich long-term hepatocyte system. Those tests which do so should begin the prevalidation and validation process.
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Organotypic Brain-slice Cultures from Adult Rats: Approaches for a Prolonged Culture Time

Eckbert Wilhelmi, Ulrich H. Schöder, Akilah Benabdallah, Frank Sieg, Jörg Breder and Klaus G. Reymann

Animal experiments are widely used in neurobiological and neuropharmacological research. Today, juvenile brain organotypic slice cultures have partially replaced in vivo experiments, but there is no adequate in vitro counterpart for the adult brain. The present study was aimed at the long-term culture of physiologically intact hippocampal slices from adult rats, by improving the conditions for preparation and culture, and the development of a new culture medium. A cerebrospinal fluid (CSF)-like medium was used, which was modified with a variety of supplements, including energy precursors, free-radical scavengers, and compounds known to inhibit neurotoxicity. The population spike amplitude (PSA) was used as a measure of viability, and amplitudes larger than 1mV indicated viable cultures. The addition of MK-801 during slice preparation improved PSA values during the first two days in vitro (DIV). Ascorbic acid and insulin prolonged the culture time up to DIV 4. FK-506 and vitamin E, alone or in combination, supported slice culture up to DIV 5. An increase in ATP, unless combined with vitamin E, and/or insulin, increased culture time up to DIV 6. Vitamins B1, B2, B12 and D2 had no effect. The modified CSF-like medium developed in this study permits the culture of adult hippocampal tissue for at least 6 days.
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Long-term, Repeated Dose In Vitro Neurotoxicity of the Glutamate Receptor Antagonist L-AP3, Demonstrated in Rat Hippocampal Slice Cultures by Using Continuous Propidium Iodide Incubation

Bjarne W. Kristensen, Morten Blaabjerg, Jens Noraberg and Jens Zimmer

Most in vitro models are only used to assess the short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2–3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100μM L-AP3, with PI (2μM) in the culture medium. Exposure to 100μM L-AP3 induced severe toxicity after 4–6 days, as shown by massive PI uptake, LDH leakage, and changes in MAP2 and GFAP immunostaining and in Nissl and Timm staining. In contrast, 10μM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist, NBQX, together with 100μM L-AP3, reduced neurodegeneration to close to control values. It is concluded that the continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies on inducible neuronal degeneration over time.
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