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Expression of a highly differentiated phenotype and hepatic functionality markers in gilthead seabream (Sparus aurata L.) long-cultured hepatocytes: first morphological and functional in vitro characterization

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Abstract

The development of primary cultures and cell lines from aquatic organisms is a valuable tool for a wide range of research activities applied to aquaculture. Despite several efforts, derivation and long-term culturing of primary hepatocytes from marine vertebrates are still rare and unsuccessful. This is the first report to fully characterize long-term cultures of primary hepatocytes from the European seabream, Sparus aurata L. (Osteichthyes, Sparidae) (SaHePs). In this new model, hepatocyte cells were long-term viable, active proliferating, and fully retained liver function up to 3 weeks. SaHePs expressed a differentiated phenotype, owing to the reacquisition of the peculiar cytoarchitecture with the complete assembly of cytoskeletal and junctional network, as shown by the production and immunolocalization of several polarity markers and cytoskeletal proteins (MDR1, ZO-2, C-CAM1, Vimentin, Cadherin, β-Tubulin, β-Catenin, β-Actin). Cytostructural analysis to identify polarized expression and bile canaliculi formation was performed by immunofluorescence and contrast phase microscopy. Long cultured SaHePs also demonstrated evidence of Albumin, α1-Antitrypsin (AAT) and α-Fetoprotein (AFP) synthesis, expression of the detoxifying metabolic enzyme cytochrome P-4501A (CYP 1A), and production of hepatocyte specific cytoskeleton proteins, such as Cytokeratin 8 (CK8) and Cytokeratin 18 (CK 18). The presence of specific markers for hepatic phenotype, detected by immunocytochemistry and Western blot analysis, is suggestive of the full maintenance of a highly differentiated phenotype and hepatic maturation. These data demonstrate that SaHePs can be long cultured without losing the hepatic functionality. This study provides a useful tool for innovative research applications in fish toxicological, pathological, and physiological studies, as one of the few hepatic, functionally active, in vitro model from marine fish.

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Acknowledgments

Authors would like to thanks the Panittica Pugliese S.p.A. for providing the experimental animals. The excellent critical reading of Dr. Marcella Narracci (Institute for Coastal Marine Environment, National Research Council, Taranto, Italy) is gratefully acknowledged. Authors would like to thanks the helpful technical support of Dr. Giovanna Calzaretti and Francesco D’Onghia.

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  1. Division of Aquaculture, Department of Public Health and Animal Science, Faculty of Veterinary Medicine, University of Bari, Str. Prov. per Casamassima Km 3, 70010, Bari, Valenzano, Italy

    Maria Pia Santacroce, Valentina Zacchino & Gerardo Centoducati

  2. Division of Veterinary Biochemistry, Department of Pharmaco-Biology, Faculty of Veterinary Medicine, University of Bari, Bari, Italy

    Elisabetta Casalino & Elisabetta Merra

  3. Division of Pharmacology and Toxicology, Department of Public Health and Animal Science, Faculty of Veterinary Medicine, University of Bari, Bari, Italy

    Giuseppe Crescenzo

  4. Division of Animal Science, Department of Public Health and Animal Science, Faculty of Veterinary Medicine, University of Bari, Bari, Italy

    Alessandra Tateo & Pasquale De Palo

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  1. Maria Pia Santacroce
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  2. Valentina Zacchino
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  3. Elisabetta Casalino
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  4. Elisabetta Merra
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Correspondence to Maria Pia Santacroce.

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Santacroce, M.P., Zacchino, V., Casalino, E. et al. Expression of a highly differentiated phenotype and hepatic functionality markers in gilthead seabream (Sparus aurata L.) long-cultured hepatocytes: first morphological and functional in vitro characterization. Rev Fish Biol Fisheries 21, 571–590 (2011). https://doi.org/10.1007/s11160-010-9181-7

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