Use of a low-speed,iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations |
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Authors: | Bill L. Kreamer Jeffrey L. Staecker Norimasa Sawada Gerald L. Sattler M. T. Stephen Hsia Henry C. Pitot |
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Affiliation: | (1) Environmental Toxicology Center and Department of Entomology, University of Wisconsin, 53706 Madison, Wisconsin;(2) McArdle Laboratory for Cancer Research, University of Wisconsin, 53706 Madison, Wisconsin;(3) 237 Russell Laboratories, University of Wisconsin, 1630 Linden Drive, 53706 Madison, Wisconsin |
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Abstract: | Summary A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 ×g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene. This work was supported in part by the National Institutes of Health Biomedical Research Support Program, and National Institute of Environmental Health Services grant (ES-01737) awarded to M.T.S.H., and by National Cancer Institute grants CA-017175, CA-09135, CA-22484 awarded to H.C.P.N.S. was supported by a Cancer Research Campaign Grant (U. K.) through the International Union Against Cancer. This work was presented in part at the 24th Annual Meeting of the Society of Toxicology, 18–22 March 1985, San Diego, CA. |
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Keywords: | isolated rat hepatocytes percoll viability DNA repair cytochrome P-450 tyrosine aminotransferase |
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