Cultured trout gill epithelia enriched in pavement

Authors: Fernando Galvez Tommy Tsui Chris M Wood

Publish Date: 2008/09/23

Volume: 44, Issue: 10, Pages: 415-425

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Abstract

The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led in recent years to the development of cultured gill epithelia on semipermeable supports To date their minimal capacity to actively transport ions has limited their utility as ionoregulatory models The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement PV cells or a mixed population of PV cells and mitochondriarich MR cells from the gills of adult rainbow trout Although the cell culture approach is similar to the doubleseeded insert DSI technique described previously it makes use of Percoll density centrifugation to first separate populations of PV and MR cells which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types Based on rhodamine staining the MR cellrich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations In general MR cellrich epithelia developed extremely high transepithelial resistances TER 30 kΩ cm2 and positive transepithelial potentials TEP under symmetrical conditions ie L15 medium on both apical and basolateral sides Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations although MR cellrich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions Measurement of unidirectional Na+ fluxes and application of the Ussing flux ratio criterion demonstrated active Na+ uptake in PV cellrich and MR cellrich epithelia under both symmetrical and asymmetrical conditions In comparison Ca2+ uptake and Na+/K+ATPase activity were significantly elevated in MR cellrich preparations relative to the traditional DSI or PV cellrich cultures under symmetrical conditions This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells thereby illuminating the ionoregulatory functions of the two cell types

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