Open AccessDevelopment of a rainbow trout intestinal epithelial cell line and its response to lipopolysaccharide
Author(s) -
KAWANO A.,
HAIDUK C.,
SCHIRMER K.,
HANNER R.,
LEE L.E.J.,
DIXON B.,
BOLS N.C.
Publication year - 2011
Publication title -
aquaculture nutrition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.941
H-Index - 79
eISSN - 1365-2095
pISSN - 1353-5773
DOI - 10.1111/j.1365-2095.2010.00757.x
Subject(s) - viability assay , biology , glutamine , fetal bovine serum , cell culture , lipopolysaccharide , alkaline phosphatase , rainbow trout , microbiology and biotechnology , ec50 , biochemistry , cell , in vitro , immunology , enzyme , fish <actinopterygii> , genetics , fishery , amino acid
A cell line, RTgutGC, was developed from the intestine of Oncorhynchus mykiss . RTgutGC has an epithelial‐like shape, been passaged over 100 times, and cryopreserved successfully. A rainbow trout origin was confirmed by sequencing a 652 bp region of the mitochondrial cytochrome c oxidase I gene. RTgutGC is grown routinely in Leibovitz’s L15 without glutamine supplemented with 10% fetal bovine serum (FBS). Cell viability was evaluated with Alamar blue (AB) for metabolic activity and carboxyfluorescein diacetate acetoxymethyl ester (CFDA AM) for membrane integrity. Viability was unchanged by lipopolysaccharide (LPS) for cultures in FBS. For cultures at low cell densities in L15 without FBS or glutamine, cell viability declined in a LPS dose‐dependent manner, allowing calculation of the concentration causing a 50% decline in viability (EC 50 ). When glutamine was present, the EC 50 was increased for both AB and CFDA AM. As the cell density increased, LPS became much less cytotoxic and no EC 50 could be calculated for very confluent cultures. Only high‐density cultures had alkaline phosphatase (AP) activity. Thus, glutamine and possibly AP protect against LPS cytotoxicity. RTgutGC should be a useful in vitro tool for studying problems of nutrition and gastrointestinal health in fish.