Assessment of Initial Damage and Recovery Following Exposure of MDCK Cells to an Irritant

Abstract

The ability of Madin–Darby canine kidney cells (MDCK ) to form cell tight-junctions and orientate correctly on porous membranes has been exploited to model corneal barrier function. While the ability to monitor recovery profiles has not yet been included in a prevalidation trial, its inclusion facilitates the prediction of potential adverse reactions. Combining a viability assay (Alamar blue reduction) with the fluorescein leakage assay, the chemical effects on cell membrane and adhesion molecules can be distinguished. The dose–response curves obtained with Tween 20, isopropanol and benzalkonium chloride indicate that Tween 20 at 200 mg/ml caused a 50% decline in Alamar blue reduction and fluorescein retention, while 100 mg isopropanol/ml and 0.1 mg benzalkonium chloride/ml cause similar effects. The more severe the initial damage the longer the recovery period. If a 20% increase in fluorescein leakage gave recovery in 48 hours, 50% gave marginal recovery in 96 hours. Comparable effects were noted for the restoration of the ability to reduce the Alamar blue dye. Resolution of the damaging effects of benzalkonium chloride and isopropanol took longer than for Tween 20, as is the case in the rabbit in vivo.