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

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 prim...

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Bibliographic Details
Published in:Ecotoxicology (London) 2012-11, Vol.21 (8), p.2419-2429
Main Authors: Baron, Matthew G., Purcell, Wendy M., Jackson, Simon K., Owen, Stewart F., Jha, Awadhesh N.
Format: Article
Language:English
Subjects:
Albumins - metabolism
Animal behavior
Animals
Bioaccumulation
Cell Culture Techniques - methods
Cell Survival
Dehydrogenase
Earth and Environmental Science
Ecology
Ecotoxicology
Ecotoxicology - methods
Energy Metabolism
Environment
Environmental Management
Environmental Monitoring - methods
Ethylenediaminetetraacetic acid
Glucose - metabolism
Hepatocytes - cytology
L-Lactate Dehydrogenase - metabolism
Liver - anatomy & histology
Liver - cytology
Mammals
Methods
Microscopy, Electron, Scanning
Microscopy, Fluorescence
Oncorhynchus mykiss - anatomy & histology
Oncorhynchus mykiss - physiology
Perfusion
Salmon
Spheroids, Cellular - cytology
Technical Note
Trout
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Summary: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  
ISSN:0963-9292
1573-3017
DOI:10.1007/s10646-012-0965-5