Comparative analysis of 3D and 2D in vitro models of the permanent fish liver cell line RTL-W1: Metabolic capabilities and responses to xenobiotics

Abstract

In vitro models based on permanent fish liver cell lines have proven to be versatile tools for examining chemical biotransformation and toxicity. However, their in vivo relevance remains uncertain due to their potentially de-differentiated phenotype. Here, we investigate whether a 3D cell culture environment can restore hepatocyte-like properties of the Rainbow trout liver cell line RTL-W1. Utilizing ultralow attachment (ULA) microwell plates, we achieved controlled sizing and extended culture (3 weeks) of spheroidal aggregate cultures (spheroids). RTL-W1 cells within the spheroids remained viable and metabolically active, as confirmed by the CellTiter-Glo 3D assay. Transmission electron microscopy revealed that spheroids exhibit tissue-like arrangements, such as interdigitations, cell-cell junctions, and endo- or exocytic activity at the cell-cell interface. They also displayed ultrastructural characteristics typical of metabolically active cells/hepatocytes, including abundant endoplasmic reticulum (ER), Golgi apparatus, and mitochondria. RT-qPCR analysis showed upregulation of genes involved in xenobiotic and endogenous (lipid) metabolism in 3D cultures over time. Notably, for several genes, especially cyp1a, expression levels were significantly higher in spheroids than in monolayers cultured for the same duration. This was corroborated at the enzyme level by increased Cyp1a-dependent catalytic activity (EROD). Interestingly, increased Cyp1a expression did not lead to heightened susceptibility to benzo[a]pyrene toxicity, which requires bioactivation. However, RTL-W1 3D and 2D cell cultures exhibited differential susceptibility to toxicity from other model chemicals, such as the surfactant SDS and the metal copper (Cu). These findings support the hypothesis that RTL-W1 cells can re-differentiate to a hepatocyte-like phenotype when cultured in a 3D configuration and may exhibit distinct biological responses upon exposure to xenobiotics. Overall, this study advances our understanding of the potential of cell line-derived 3D in vitro models for research and providing more physiologically relevant data for regulatory contexts.

Keywords: 3D cell culture; Bioaccumulation; Ecotoxicology; Fish; Hepatocyte; NAM; Spheroid.