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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 \u00b1 1 \u00b0C 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 &lt; 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 \u00b1 1.9 \u00d7 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.<\/jats:p>","DOI":"10.3390\/ijerph18041380","type":"journal-article","created":{"date-parts":[[2021,2,3]],"date-time":"2021-02-03T11:54:31Z","timestamp":1612353271000},"page":"1380","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion"],"prefix":"10.3390","volume":"18","author":[{"given":"Neusa","family":"Figueiredo","sequence":"first","affiliation":[{"name":"MARE\u2014Marine and Environmental Sciences Centre, Departamento de Ci\u00eancias e Engenharia do Ambiente, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal"}]},{"given":"Beatriz","family":"Matos","sequence":"additional","affiliation":[{"name":"MARE\u2014Marine and Environmental Sciences Centre, Departamento de Ci\u00eancias e Engenharia do Ambiente, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1571-0366","authenticated-orcid":false,"given":"M\u00e1rio","family":"Diniz","sequence":"additional","affiliation":[{"name":"UCIBIO\u2014Applied Molecular Biosciences Unit, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0466-4934","authenticated-orcid":false,"given":"Vasco","family":"Branco","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]},{"given":"Marta","family":"Martins","sequence":"additional","affiliation":[{"name":"MARE\u2014Marine and Environmental Sciences Centre, Departamento de Ci\u00eancias e Engenharia do Ambiente, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal"},{"name":"UCIBIO\u2014Applied Molecular Biosciences Unit, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,2,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1016\/j.marpolbul.2018.04.025","article-title":"Microplastics on the Portuguese coast","volume":"131","author":"Antunes","year":"2018","journal-title":"Mar. 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