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Article

Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion

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MARE—Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia do Ambiente, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal
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UCIBIO—Applied Molecular Biosciences Unit, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal
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Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal
*
Author to whom correspondence should be addressed.
Academic Editor: Paul B. Tchounwou
Int. J. Environ. Res. Public Health 2021, 18(4), 1380; https://0-doi-org.brum.beds.ac.uk/10.3390/ijerph18041380
Received: 31 December 2020 / Revised: 29 January 2021 / Accepted: 31 January 2021 / Published: 3 February 2021
(This article belongs to the Section Environmental Health)
Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs. View Full-Text
Keywords: primary hepatocytes; in vitro assays; Sparus aurata primary hepatocytes; in vitro assays; Sparus aurata
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MDPI and ACS Style

Figueiredo, N.; Matos, B.; Diniz, M.; Branco, V.; Martins, M. Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion. Int. J. Environ. Res. Public Health 2021, 18, 1380. https://0-doi-org.brum.beds.ac.uk/10.3390/ijerph18041380

AMA Style

Figueiredo N, Matos B, Diniz M, Branco V, Martins M. Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion. International Journal of Environmental Research and Public Health. 2021; 18(4):1380. https://0-doi-org.brum.beds.ac.uk/10.3390/ijerph18041380

Chicago/Turabian Style

Figueiredo, Neusa, Beatriz Matos, Mário Diniz, Vasco Branco, and Marta Martins. 2021. "Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion" International Journal of Environmental Research and Public Health 18, no. 4: 1380. https://0-doi-org.brum.beds.ac.uk/10.3390/ijerph18041380

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