A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC

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.

Fish-gut-on-chip: development of a microfluidic bioreactor to study the role of the fish intestinein vitro

“…15 In contrast, TEER values obtained for RTgutGC monolayer exposed to high shear stress of 0.06 dyne per cm 2 were again comparable to values obtained for static and low shear stress (0.002 dyne per cm 2 ) conditions. Overall, the resistance values of RTgutGC monolayer cultured under static conditions in the microfluidic bioreactor are in accordance with previously reported values obtained in static cell culture inserts 8,9 and on ultrathin alumina membranes. 13 For freshwater adapted Atlantic salmon (Salmo salar), TEER values between 30 and 150 Ω cm 2 have been reported from isolated gut-sac preparations, which are composed of the epithelial cell layer and the underlying fibroblasts; 32 thus RTgutGC cell cultures under static and flow conditions generally closely reflect the in vivo TEER in salmonids.…”

“…These results are consistent with previous studies demonstrating that RTgutGC remain in monolayer for at least 14 days when seeded at a similar density on permeable membranes. 8,13 In the fish-gut-on-chip device, RTgutGC cells possessed a height of ∼6 μm at day 1. During the culture period of 9 days, cells increased only marginally in height to 7-9 μm, which was independent of flow and co-culturing (Fig.…”

“…The RTgutGC cell line, derived from the epithelium of the intestine of rainbow trout, 7 has previously been implemented in commercial trans-well inserts, allowing to study the physiological and toxicological response of the cells e.g. during simulated seawater adaptation and silver exposure, 8 as well as intestinal uptake of potential hazardous nanomaterial 9 and hydrophobic chemicals. 10 Yet, commercial inserts have limitations, such as low permeability of the polymeric cell culture membrane and limited visibility of cells.…”

Nanomaterials in the environment: Behavior, fate, bioavailability, and effects—An updated review

“…To accomplish NM transport studies in cells from nonmammalian species, a recently established fish epithelial barrier system (Minghetti et al 2017) based on a rainbow trout (Oncorhynchus mykiss) intestinal cell line has been used. The RTgutGC cell line (Kawano et al 2011) was adopted to allow study of particle translocation and toxicity in the intestine of fish (Geppert et al 2016).…”

“…The RTgutGC cell line (Kawano et al 2011) was adopted to allow study of particle translocation and toxicity in the intestine of fish (Geppert et al 2016). The RTgutGC intestinal model forms a leaky epithelium, which is in accordance with the fish intestinal epithelium in vivo (Geppert et al 2016;Minghetti et al 2017). It nevertheless formed an effective barrier for polystyrene NMs (50 nm nominal size): after 24 h of exposure, approximately 80% of the particles remained in the apical compartment, and 9 to 16% were associated with the cells, leaving <10% permeation to the basolateral side.…”