Abstract
The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na+/K+-ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion.
Highlights
The fish intestinal epithelium is an important environment-organism interface whose physiological functions include nutrient transport, osmoregulation and protection from environmental stressors, such as toxicants (Grosell et al 2011)
When presenting the first characterization of the RTgutGC cell line, Kawano et al (2011) proposed that this intestinal cell line may have the potential to become the fish equivalent of the Caco-2 cell line, which is intensively used as human model of pharmacokinetics and RTgutGC cell density when cultured on the permeable membranes over time. a Evolution of transepithelial electrical resistance (TEER) in inserts of two different sizes: 0.33 and 1.13 cm2
Our results are in support of this proposal; in contrast to the Caco-2 cell line, which was derived from a human colon carcinoma, RTgutGC cells were initiated from a healthy rainbow trout
Summary
The fish intestinal epithelium is an important environment-organism interface whose physiological functions include nutrient transport, osmoregulation and protection from environmental stressors, such as toxicants (Grosell et al 2011). Sundh et al (2014) and Grosell et al (2007), for example, have used molecular, biochemical and immunohistochemical approaches on salmonids to understand the role of the fish intestine during the process of acclimation to seawater These types of studies, while relying on a fully developed, functional epithelium, do not allow for controlled variation of experimental parameters on the level of the organ itself. Freshly isolated intestinal epithelial cells can be used for short term in vitro investigations Such an approach was, for example, chosen by Burke and Handy (2005) and Kwong and Niyogi (2012) to evaluate time- and concentration-dependent copper and cadmium accumulation. To provide a much more accessible, tuneable, animal-free model for mechanistic studies on polarized fish intestinal cells, we here present details of the establishment of a novel fish epithelial barrier model based on the rainbow trout gut cell line, RTgutGC (Kawano et al 2011)
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