Characterization of RTgill-W1 cells epithelial development on transwell inserts: Evaluation of osmotic and toxic challenges

Abstract

RTgill-W1 cells cannot be directly exposed to freshwater (FW) or seawater (SW) due to osmotic stress. Adjustments of exposure solutions are needed, but these might reduce the bioavailability and toxicity of pollutants. To facilitate cell polarization and allow direct exposure of water samples, cells were cultured on transwell inserts. Monolayer formation was measured by trans-epithelial electrical resistance (TEER) and an apparent permeability (Papp) assay. At 14 days both TEER and Papp indicated the lowest permeability. Cell viability showed that cells can tolerate apical FW with complete medium (L-15/FBS) in the basolateral compartment but SW reduced cell viability. However, when reference toxicants, silver nitrate and sodium dodecyl benzene sulfonate, were added no toxicity was detected. Increased osmolality in the apical side and presence of proteins indicated diffusion from the basolateral to the apical side. Thus, reduced toxicity was likely caused by complexation with media salts and amino acids. A protein and amino acid free exposure medium (L-15/ex) was applied in the basolateral compartment. However, FW exposures with basolateral L-15/ex resulted in reduced cell viability. To reduce osmotic stress, mannitol was added to apical FW maintaining basolateral L-15/ex which improved cell viability and allowed detection of silver toxicity. Finally, RTgill-W1 cells did not show normal tight junction protein (ZO-1) immunocytochemical staining, which fits with the formation of a leaky epithelium. Overall, culturing of RTgill-W1 cells on transwell inserts allowed direct exposure to mannitol FW medium but showed a reduced sensitivity to toxicants. Thus, exposure on flat bottom wells is recommended for routine toxicity testing.