Cultured gill epithelia as models for the freshwater fish gill

Abstract

We review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral "influx" of Cl(-) (and not of Na(+)). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na(+),K(+)-ATPase activity and promoting active Na(+) and Cl(-) influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na(+) uptake demonstrate that both NH(3) and NH(4)(+) diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na(+)-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested.

Primary cultured cells as sensitive in vitro model for assessment of toxicants-comparison to hepatocytes and gill epithelia

“…Importantly, TER remained unchanged upon exposure to toxicants among groups, which suggests that the integrity was not affected by toxicants. The rapid increase in TER upon apical side exposure to freshwater may have resulted from the close of the apical channel (Wood et al, 2002). TER declined gradually after long-term exposure (6-24 h), which was also reported by previous studies of freshwater rainbow trout gill epithelia.…”

“…However, the TER from freshwater tilapia in the plateau phase value was comparable to that in seawater tilapia. TER is an indicator of the tightness of epithelial cells (Wood et al, 2002). In the present study, the TER value is general around 5000-8000 cm 2 , which is believed to indicate "tight" epithelia.…”

“…Gill cells were isolated in a laminar flow hood using sterile techniques according to Wood and Pärt (1997). The double-seeded insert (DSI) epithelia were prepared following Kelly et al (2000) and Zhou et al (2005).…”

“…Pärt et al (1993) initially developed primary cultured gill epithelia in culture plates from freshwater rainbow trout (Oncorhynchus mykiss). Since then, several gill epithelia of freshwater rainbow trout that have been grown on filter "inserts" have been developed as a surrogate in vitro primary cultured model for fish gills (Wood and Pärt, 1997;Fletcher et al, 2000;Wood et al, 2003). Among these, doubleseeded insert (DSI) preparations that incorporate both pavement cells (respiratory cells) and mitochondria-rich cells (MRCs) can form two to four layers of epithelial cells with polarity.…”

Tight junction protein gene expression patterns and changes in transcript abundance during development of model fish gill epithelia

“…Primary cultured model gill epithelia provide a platform for studying the paracellular permeability characteristics of the gill epithelium (for a review see Wood et al, 2002). These models are Tight junction protein gene expression patterns and changes in transcript abundance during development of model fish gill epithelia Dennis Kolosov*, Helen Chasiotis and Scott P. Kelly simplified flat epithelia 'reconstructed' on permeable cell culture inserts using gill epithelium pavement cells (PVCs) (Avella and Ehrenfeld, 1997;Chasiotis and Kelly, 2011a;Chasiotis and Kelly, 2011b;Kelly and Wood, 2002;Wood and Pärt, 1997) or both PVCs and gill epithelium mitochondrion-rich cells (MRCs) .…”