A Step Forward in the Characterization of Primary Brown Trout Hepatocytic Spheroids as Experimental Models

SIMPLE SUMMARY: The liver is a vital organ for xenobiotic biotransformation and a prime target for drug toxicity. The search for alternative experimental liver models in fish and other species is critical to reducing the number of individuals used in animal experiments. Mammal liver spheroids have emerged as a viable in vitro model with similar morphofunctional properties to the liver. These three-dimensional (3D) structures are not well studied in fish. In this study, primary brown trout hepatocyte spheroids were characterised in biometry, histomorphology, and basal expression of a selection of target genes (metabolism and detoxification, efflux transport, and estrogenic signalling). The spheroids resembled in vivo liver features and demonstrated a morphological and functional time window stabilisation from the 12th to the 20th day in culture. The model is promising for investigating fish hepatic adaptive and toxicological responses to xenobiotics. ABSTRACT: Mammal hepatocyte spheroids have been investigated as alternative experimental models in several contexts, since three-dimensional (3D) systems have shown the potential to mimic in vivo scenarios. The description of fish hepatocyte 3D models is still minimal. This study intends to further characterize brown trout primary hepatocyte spheroids at distinct time points up to 25 days in culture. Viability, biometry, histomorphology, and basal expression of a selection of genes (metabolism and detoxification, efflux transport, and estrogenic signalling) were considered. The gene expression of whole liver samples from the same fish donor were evaluated concurrently. After 12 days in culture, the hepatocyte spheroids exhibited biometric and morphological stability. From the 12th to the 20th day in culture, the basal expression levels for most of the selected genes did not vary. The targeted mRNA levels were higher in brown trout liver samples compared to hepatocyte spheroids. Despite that, data supported that this model resembles some in vivo features. As an experimental alternative model, it showed potential to be used in a stable time window that can be exploited for exposure tests to different xenobiotics, namely, estrogenic compounds.