Regulatory and academic studies use large numbers of fish annually. The use of fish primary cells offers an in vitro alternative for the assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. Their uses however are not without limitations which includes short culture periods (i.e. longevity and loss of organ-specific functionality over time. Three-dimensional (3-D spheroid) technology is now established for in vitro mammalian toxicity studies and offers significant advantages for environmental applications in a model fish species. This thesis reports development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compares morphological, functional and biochemical status with two-dimensional (2-D) monolayer hepatocytes. The work further assesses the bio-transformation potential of developed 3-D spheroids to a range of environmentally relevant pharmaceuticals. The study suggests that mature spheroids retain some organotypic responses over-time in culture including morphological (viz., smooth outer surface, tight cell–cell contacts); functional (viz., histo-architecture; cell adhesion molecule expression) and biochemical properties (viz., protein, glucose, albumin- and enzyme levels) that is superior to conventional 2-D monolayer cells. These 3-D spheroids also demonstrate a capacity for the metabolism of environmentally-relevant pharmaceuticals that could be utilised to better understand their bio-accumulation potential in fish. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies as well as for fundamental understanding of the interaction of chemicals with biomolecules which could potentially lead to detrimental responses at different levels of biological organisation.

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