Fish hepatocyte spheroids - A powerful (though underexplored) alternative in vitro model to study hepatotoxicity

In vitro fish cell cultures are considered alternative models to in vivo toxicological studies. The two-dimensional (2D) cultures have been used in toxicity testing, but those models have well-known drawbacks, namely in culture longevity and in the maintenance of some in vivo cellular functions. In this context, three-dimensional (3D) systems are now proposed to better mimic in vivo effects. The use of 3D cultures in fish is still limited (e.g., toxicity testing, drug biotransformation and bioaccumulation studies) compared to the number of studies with mammalian cells exploring the potential of these systems. In fish, the liver spheroids have been the most used 3D model, deriving from either liver cell lines or primary cultures of hepatocytes. Because the liver is the main organ for xenobiotic detoxification, hepatocyte spheroids represent a promising alternative to test concentration-responses to xenobiotics and explore mechanistic or ecotoxicological perspectives. Evidence shows that fish hepatocytes cultured in spheroids closely resemble the in vivo counterparts, additionally having higher basal metabolic capacity than hepatocytes cultured in monolayer. This graphical review is an updated critical sum-up of data published with 3D fish hepatocytes and provides background knowledge for the upcoming studies using this model. It further addresses the culture conditions for obtaining fish hepatocyte spheroids and discusses the main factors that can influence the biometry and functionality of spheroids over time in culture and the 2D versus 3D distinct metabolic capacities.