The anti-coagulating agent heparin and native serum alter the barrier and tight junction properties of a primary cultured model fish gill epithelium.

Primary cultured gill models are useful for investigating fish gill epithelium barrier properties. Typically, gill cell culture methods utilize commercially available heterologous serum supplements rather than native serum. This is because heterologous serum is easy to obtain and facilitates the development of models that mimic the gill very well. Also, native serum (or plasma) has historically had mixed success in primary cultured fish epithelium models. This study examined the effects of the blood anti-coagulating agent heparin and native serum on the barrier properties of a primary cultured trout (Oncorhynchus mykiss) gill model. Development of epithelia in the presence of heparin decreased transepithelial electrical resistance (TER) and increased paracellular movement of [3H]polyethylene glycol (MW 400 Da, PEG-400). In association, transcript abundance of genes encoding tight junction (TJ) proteins tricellulin, zonula occludens-1, cingulin, and claudin (cldn) -1, -8b, -8c, -8d, -12, -23a, -27b, -28b, and -29a decreased, while cldn30 and -32a mRNA abundance increased. Acute treatment with heparin for 24 h had a qualitatively similar impact on TER and paracellular permeability, but a less pronounced impact on TJ-associated genes. In contrast, replacing 10% fetal bovine serum with 10% rainbow trout serum (prepared without an anti-coagulating agent) increased TER and reduced paracellular [3H]PEG-400 permeability. This was associated with increased transcript abundance of ocln, cldn8b, -8d, -27b and -28b as well as increased protein abundance of Cldn-8d. Data suggest that native extracellular fluid can enhance model trout gill barrier properties, but that anti-coagulating agents should be evaluated to establish if they have deleterious effects.