Optimisation of three-dimensional cultures of the RTL-W1 liver cell line to model polycyclic aromatic hydrocarbon effects on liver detoxification

Climate change-induced stress and increased aquatic pollution, including persistent pollutants such as polycyclic aromatic hydrocarbons (PAHs), disrupt biological functions. Although 3D cell culture systems can be used to better understand these effects, their use in fish studies is currently limited. In this vein, developing and characterising new models is of extreme importance, as these may help to disclose pollutant effects on temperature increase scenarios.

To generate spheroids from the rainbow trout liver-derived RTL-W1 cell line, four cell densities — 10,000, 20,000, 40,000 and 60,000 cells per well — were cultured in 96-well ultra-low attachment (ULA) plates at 18 ºC, with or without centrifugation after seeding. Over 18 days in culture, the spheroids were assessed for viability (using alamar blue and lactate dehydrogenase (LDH) assays), biometry (diameter, area and sphericity) and morphology (optical and electron microscopy). Protein expression of cytochrome P450 (CYP) 1A was assessed by immunocytochemistry (ICC) at 10, 14 and 18 days in culture. To determine its suitability for ecotoxicology research, spheroids (60,000 cells) were exposed from the 10^th to the 14^th day in culture to two concentrations of the model PAH benzo(k)fluoranthene (BkF), at 18 and 23 ºC. CYP1A immunolabeling and mRNA expression were evaluated after the exposures.

In all culture densities, spheroids' diameter and area decreased over time, while sphericity and viability increased. The biometric parameters remained stable from the 10^th day onward. Centrifugation did not impact the spheroids’ formation dynamics. Morphological analysis showed unscathed cells and organelle content compatible with hepatocytic differentiation. Spheroids expressed CYP1A in ICC, denoting its functionality. BkF-treated spheroids exhibited increases in both ICC and mRNA expression, but there were no variations in gene expression across temperatures.

The generated spheroids appear to be a promising alternative model for studying PAHs and warming effects on fish liver detoxification.