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Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes

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Abstract

The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1–5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell–cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6–8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.

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Acknowledgments

The authors would like to thank Dr. Jinsheng Xu (UWE, Bristol) for his assistance with spheroid culture and Mrs. Lynne Cooper (Plymouth University) for her expertise in primary tissue culture. Mr. Gareth Readman (Brixham Environmental Laboratory) and Mr. Ben Eynon (Plymouth University) are thanked for their help with fish husbandry and Dr. Roy Moate (Plymouth University) for his expertise in electron microscopy. The authors would also like to thank Prof. Lucy Lee (Wilfrid Laurier University, Canada) for her kind donation of RTL-W1 cells. This study was funded by a Biotechnology and Biological Sciences Research Council (BBSRC)/AstraZeneca Case Award to MB.

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  1. School of Biomedical and Biological Sciences, Plymouth University, Drake Circus, Plymouth, Devon, PL4 8AA, UK

    Matthew G. Baron, Wendy M. Purcell, Simon K. Jackson & Awadhesh N. Jha

  2. AstraZeneca Safety Health and Environment, Brixham Environmental Laboratory, Brixham, Devon, TQ5 8BA, UK

    Matthew G. Baron & Stewart F. Owen

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Correspondence to Awadhesh N. Jha.

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Baron, M.G., Purcell, W.M., Jackson, S.K. et al. Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes. Ecotoxicology 21, 2419–2429 (2012). https://doi.org/10.1007/s10646-012-0965-5

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