Cultured branchial epithelia from freshwater fish gills

Abstract

We have developed a method for the primary culture of gill epithelial cells from freshwater rainbow trout on permeable supports, polyethylene terephthalate membranes ('filter inserts'). Primary cultures of gill cells (6-9 days in Leibowitz L-15 culture medium plus foetal bovine serum and glutamine) are trypsinized and the cells seeded onto the inserts. After 6 days of growth with L-15 medium on both surfaces (approximately isotonic to trout plasma), the cells form a tight epithelium as judged from a progressive rise in transepithelial resistance which reaches a stable plateau for a further 6 days, as long as L-15 exposure is continued on both surfaces. The cultured epithelium (approximately 8 µm thick) typically consists of 2-4 overlapping cell layers organized as in the lamellae in vivo, with large intercellular spaces, multiple desmosomes and putative tight junctions. The cells appear to be exclusively pavement-type cells with an apical surface glycocalyx, an abundance of rough endoplasmic reticulum, no selective DASPEI staining and relatively few mitochondria. Transepithelial resistance (approximately 3.5 k cm2), permeability to a paracellular marker (polyethylene glycol-4000; 0.17x10(-6) cm s-1) and unidirectional flux of Na+ and Cl- (approximately 300 nmol cm-2 h-1) all appear realistic because they compare well with in vivo values; net fluxes of Na+ and Cl- are zero. The preparation acidifies the apical medium, which accumulates a greater concentration of ammonia. Upon exposure to apical freshwater, resistance increases six- to elevenfold and a basolateral-negative transepithelial potential (TEP) develops as in vivo. These responses occur even when mannitol is used to prevent changes in apical osmotic pressure. Net Na+ and Cl- loss rates are low over the first 12 h (-125 nmol cm-2 h-1) but increase substantially by 48 h. The elevated resistance and negative TEP gradually attenuate but remain significantly higher than pre-exposure values after 48 h of apical freshwater exposure. The preparation may provide a valuable new tool for characterizing some of the mechanisms of active and passive ion transport in the pavement cells of the freshwater gill.