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.
Keywords: Chronic exposure; Ecotoxicology; Environmental risk assessment; Fish gill; In vitro; Oncorhynchus mykiss.