Gaseous oxide toxicity evaluated with cell monolayers on collagen-coated, gas-permeable teflon membranes

A system was developed to evaluate the cytotoxic potential of gaseous oxides in vitro. Target cells were MRC-5 human lung fibroblasts cultivated as monolayers on gas-permeable , FEP-Teflon membranes. Membranes were secured in Chamber/Dishes with a 25 mm diameter well. To promote attachment of fibroblasts to the membranes, the latter were incubated in collagen ( Vitrogen ) solutions for 10 min prior to plating the cells. The collagen pretreatment was significantly more effective than poly-L-lysine, fetal calf serum, polybrene and bovine serum albumin. Several types (mouse and calf) of acid-soluble and alcohol-soluble collagen fractions were evaluated, and all of them promoted cell attachment with equivalent efficiency. Cells on membranes were exposed to gases in a Plexiglass chamber with a gas flow of 2L/min. Sulfur dioxide caused a marked loss in cell viability (as indicated by ATP content of the monolayer) after 30 min exposure to 0.01% and 0.005%. A level of 0.001% did not affect viability, and none of the levels tested caused a sloughing of the monolayer after 90 min. Nitrogen dioxide induced a more modest drop in cell viability after 30 min exposure to 0.1%, while 0.005% and 0.05% were nontoxic. No cell sloughing occurred with NO2 exposures, and exposures to CO2 at levels of 20% for 90 min were nontoxic. This system, with cell culture monolayers on gas-permeable Teflon membranes, is simple and convenient. As such, it has potential application to cytotoxicity evaluations with numerous gases.