In vitro Methodologies for Environmental Risk Assessment: Comparison between methods for the isolation of marine fish primary hepatocytes

Abstract

Interest of in vitro methods has grown exponentially throughout the years especially due to the ethical problems surrounding the use of animals in in vivo studies. Cell-based assays for the isolation of hepatocytes using enzymatic methods have been credited in fish toxicology and play a substantial role in the propagation of in vitro methodologies, having been implemented with various degrees of success to a variety of fish species, especially in freshwater species. Isolated hepatocytes provide an optimal system to study the role of hepatic metabolism in the activation of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs). For these reasons, the present work focused on the optimization of the isolation of two marine fish hepatocytes (P. maxima and S. aurata) using two enzymatic methods of isolation, pancreatin digestion and 2-step collagenase perfusion, for posterior validation in cytotoxicity testing. The two main parameters studied were cell viability and yield after isolation. Cell viability was assessed via the Trypan blue exclusion test and cell yield was counted with the use of a hemocytometer. The pancreatin method accounted for better overall cell yield and also produced the highest viability out of all experiments. Pancreatin digestion isolations also produced purer cultures, largely with 100% viability, which was made possible by way of modifications made during isolations, such as the use of histopaque for blood cell separation and addition of another smaller mesh filter (60 μm) to achieve better purity and fewer cell debris, respectively. A cytotoxicity assay was also performed using the MTT colorimetric assay to access cell viability after contamination with Phenanthrene and Benzo[b]fluoranthene, two polycyclic aromatic hydrocarbons (PAHs) of interest for cytotoxicity studies. Viabilities after contamination with both compounds only exhibited small variability demonstrating no dose-response curve, likely due to factors such as intra-species individual variability and the precipitation of contaminants in the highest concentrations, which might have affected absorbance readings and could cause misleading cell viabilities. Therefore, we call for further studies to access the cytotoxic effect on P. maxima and S. aurata by Phe and B[b]f. In conclusion, the research showed that the best method for isolation was the pancreatin digestion and the marine fish primary hepatocyte cultures obtained are suitable for the application in cytotoxicity assays.