Development of an in vitro model of fish skin and gill

Rainbow trout explants of skin and gill were cultured in Dulbeccoʹs modified Eagleʹs medium containing 5% heat-inactivated fetal bovine serum. Trout skin pieces (0.5–1 cm) or whole gill arches were placed in a controlled atmospheric chamber, gassed with a mixture of 45% O2, 5% CO2 and 50% N2, and incubated on a rocking platform at 13°C. Tissues were fixed at intervals of up to 30 days for light and electron microscopy. In skin cultures, the epidermis including scales, mucous cells and dermis remained normal for at least 30 days by which time epithelial outgrowth had occurred along the edges of the explant and onto the culture dish forming a monolayer. Similar normally appearing gill epithelium was noted including preservation of epithelial cells, chloride cells and pillar cells in the central stroma. Cultures were exposed to 1 mM or 10 μm HgCl2 for up to 6 h. These cultures showed marked changes in the epithelium including swelling of cytosol, endoplasmic reticulum, and mitochondria and nuclear lysis. By 6 h, many cells were necrotic with flocculent intramatrical densities in mitochondria and interruption in plasmalemmal permeability. In many gill filaments, only the pillar cells and capillaries remained intact. These studies show that skin and gill can be successfully cultured and that they remain essentially normal for up to 30 days. In addition, skin explants can serve as an important way to initiate monolayer cultures. They also show that gill and skin show responses to environmental/toxic injury that lead to cell death at higher doses.