Trout hepatocyte culture: Isolation and primary culture |
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Authors: | James E Klaunig Randall J Ruch Peter J Goldblatt |
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Institution: | (1) Department of Pathology, Medical College of Ohio, 3000 Arlington Avenue, 43699 Toledo, OH |
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Abstract: | Summary Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×106 liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes
in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations
of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the
incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep
450 activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d
of culture. Culture temperature also influenced thep
450 activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated
hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes
between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and
the appearance of bundles of intermediate filaments also were observed with increased time in culture.
This work was supported in part by an American Cancer Society Grant (Ohio Division, Inc.) and an NIH Biomedical Research Support
Grant 5507RR05700010. |
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Keywords: | trout hepatocyte culture teleost liver |
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