Cultured branchial epithelia from freshwater fish gills

Chris M. Wood, Peter Pärt

Research output: Contribution to journalArticle

102 Citations (Scopus)

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 kΩ cm2), permeability to a paracellular marker (polyethylene glycol-4000; 0.17 x 10-6 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 Clare zero. The preparation acidities 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.

Original languageEnglish
Pages (from-to)1047-1059
Number of pages13
JournalJournal of Experimental Biology
Volume200
Issue number6
StatePublished - Mar 1 1997

Fingerprint

Fresh Water
freshwater fish
gills
Fishes
epithelium
Epithelium
fish
pavement
Ion Transport
cells
mitochondrion
Glycocalyx
Desmosomes
Polyethylene Terephthalates
Primary Cell Culture
Trout
Rough Endoplasmic Reticulum
Tight Junctions
Oncorhynchus mykiss
rainbow

Keywords

  • cell culture
  • epithelial cells
  • filter inserts
  • gills
  • ionic fluxes
  • Oncorhynchus mykiss
  • rainbow trout
  • transepithelial potential
  • transepithelial resistance

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Agricultural and Biological Sciences (miscellaneous)

Cite this

Cultured branchial epithelia from freshwater fish gills. / Wood, Chris M.; Pärt, Peter.

In: Journal of Experimental Biology, Vol. 200, No. 6, 01.03.1997, p. 1047-1059.

Research output: Contribution to journalArticle

Wood, CM & Pärt, P 1997, 'Cultured branchial epithelia from freshwater fish gills', Journal of Experimental Biology, vol. 200, no. 6, pp. 1047-1059.
Wood, Chris M. ; Pärt, Peter. / Cultured branchial epithelia from freshwater fish gills. In: Journal of Experimental Biology. 1997 ; Vol. 200, No. 6. pp. 1047-1059.
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