An in vitro system to screen for diarrheagenic chemicals

We examined an in vitro system to screen for diarrheagenic chemicals using an established intestinal cell line (T₈₄ human colonic carcinoma). The cells were grown on Millicell-PCF (polycarbonate membrane) wells. The cells were seeded at approximately 5 × 10₆ cells/30mm well and incubated for 9–11 days in a 5% C0₂ incubator saturated with water at 37°C. The culture medium was a 1:1 mixture of Ham's F12 and Dulbecco's MEM with 5% fetal bovine serum and 25 pglml gentamicin sulfate. The well containing cells was removed from the incubator and mounted in a modified Ussing chamber for measurement of shortcircuit current (I_sc). Chemical-induced increases in Psc are usually indicative of electrogenic epithelial Cl^– secretion, which is associated with diarrheagenic effects in animals and humans. T₈₄ cells grown on Millicell-PCF membrane responded with an increase in Isc after basolateral addition of the cholinergic (muscarznic) agonist carbachol, prostaglandin E₂, 16,16-dimethylprostaglandin E₂, and forskolin, while non-diarrheagenic prostaglandin D₂ did not affect I_sc. Based on our results, this in vitro system has the potential to be adapted as a rapid screen for detecting diarrheagenic chemicals.Abbreviations dmPGE₂ 16,16-dimethylPGE₂ - EC₅₀ 50% of maximum effective concentration - EDTA ethylenediaminetetraacetate - I_SC short-circuit current - PGD₂ prostaglandin D₂ - PGE₂ prostaglandin E₂ - PD potential difference - R_T transepithelial resistance