Investigations to extend viability of a rainbow trout primary gill cell culture
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The primary culture of fish gill cells can provide functional, cell diverse, model in vitro platforms able to tolerate an aqueous exposure analogous to in vivo tissues. The utility of such models could be extended to a variety of longer term exposure scenarios if a method could be established to extend culture viability when exposed to water for longer periods. Here we report findings of a series of experiments to establish increased longevity, as monitored by culture transepithelial electrical resistance (TEER) and concurrent histological developments. Experimental cultures improved TEER during apical freshwater exposure for a mean of twelve days, compared to previous viabilities of up to 3 days. Cultures with larger surface areas and the use of trout serum rather than foetal bovine serum (FBS) contributed to the improvement, while perfusion of the intact gill prior to cell harvest resulted in a significantly faster preparation. Detailed scanning electron microscopy analysis of cultures revealed diverse surface structures that changed with culture age. Cultures grown on membranes with an increased porosity, collagen coating or 3D structure were of no benefit compared to standard membranes. Increased culture longevity, achieved in this study and reported for the first time, is a significant breakthrough and opens up a variety of future experimentation that has previously not been possible. The extended viability facilitates exploration of in vitro chronic or pulse-exposure test paradigms, longer term physiological and environmental monitoring studies and the potential for interactive co-culture with other organoid micro-tissues.
KeywordsIn vitro Oncorhynchus mykiss Fish gill Ecotoxicology Chronic exposure Environmental risk assessment
The authors would like to thank Ben Eynon, Glenn Harper, Lynne Cooper, and Andrew Atfield (University of Plymouth) for technical support.
This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) Research Grant IPA (BB/L01016X/1), co-funded by the AstraZeneca Global Safety, Health and Environment research programme, to ANJ supporting RJM and MGB. AstraZeneca provided support in the form of salary for SFO and grant to ANJ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The SFO work represents an AstraZeneca contribution in kind to the Innovative Medicines Initiative (IMI) under grant agreement no.115735—iPiE: Intelligent led assessment of Pharmaceuticals in the Environment; resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2015-2018) and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies’ in kind contribution.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
All applicable national, and/or institutional guidelines for the care and use of animals (fish) were followed.
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