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

Neusa Figueiredo,Marta Martins,Vasco Branco,Beatriz Matos,Mário Diniz

doi:10.3390/ijerph18041380

Abstract

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.

Highlights

Aquatic ecosystems, from coastline to open ocean, are widely impacted by a countless number of xenobiotics (e.g., heavy metals, persistent organic pollutants (POP), pharmaceuticals, endocrine-disrupting chemicals (EDCs), and plastic wastes and their related chemicals (e.g., BPA, phthalates)) [1,2,3,4,5,6,7,8,9,10]
The cell yield obtained from S. aurata liver ranged from 1.04 × 107 to 2.47 × 107 for the two-step collagenase perfusion method and 3.3 × 107 to 7.31 × 107 for the pancreatin digestion method (Table 1), and the cell viability ranged from 70.7 to 72.8% for the two-step collagenase perfusion method and 95.1% to 100% for the pancreatin digestion method (Table 1)
The results showed a significant increase of Ethoxyresorufin O-Deethylase (EROD) activity at This assessment was performed after 24 (T24) and T48, relative to control cells (p < 0.05)

Summary

Introduction

From coastline to open ocean, are widely impacted by a countless number of xenobiotics (e.g., heavy metals, persistent organic pollutants (POP), pharmaceuticals (e.g., triclosan), endocrine-disrupting chemicals (EDCs), and plastic wastes and their related chemicals (e.g., BPA, phthalates)) [1,2,3,4,5,6,7,8,9,10]. The presence of these xenobiotics in both water and sediments eventually results in ecotoxicological effects in aquatic organisms, from algae to fish [3,5,6].

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