Brown trout primary hepatocyte spheroids – characterization and applications in endocrinology and beyond

Abstract

Three-dimensional (3D) culture models of mammalian hepatocytes have been shown to replicate in vivo liver features accurately. The use of these 3D models avoids the constraints of an in vivo assay while sustaining the morphology and functionality of the hepatocytes throughout several days in culture. The feasibility of generating hepatic spheroids from other species, including fish, seems promising but has received less attention than in mammal models. Primary brown trout hepatocyte spheroids were first obtained by our research group from juvenile fish and cultured for over 30 days under orbital shaking. Optimization assays showed that these spheroids achieved biometric, morphological and basal gene expression stability from day 12 to day 20. This multiparametric characterization allowed the selection of a temporal window of culture days, which is being explored in several assays. The 3D primary hepatocyte spheroids were exposed to estrogens (e.g., 17α-ethinylestradiol) at different concentrations. The effect of increasing temperature was also evaluated in this model by maintaining cultures at 18°C and 21°C. Data collected include microscopy analysis, target gene expression of estrogenic and lipid pathways, and cellular biochemistry. The outputs indicate that hepatic spheroids are metabolically active and respond to different hormonal and temperature stimuli. The 3D model developed from primary brown trout hepatocytes seems to be a viable alternative for studying hepatic disruptions caused by (at least) estrogenic compounds and varied temperatures.