Evaluation of tissue culture insert membrane compatibility in the fluorescein leakage assay

The Fluorescein Leakage (FL) test, a short-term in vitro assay measuring damage on exposure to eye irritants to a transepithelial barrier permeability system, has been assessed as an alternative to the Draize rabbit eye irritation assay in the EU/Home Office international validation trial. The assay is based on the degree of leakage of sodium fluorescein through a confluent layer of Madin-Darby canine kidney epithelial (MDCK) cells grown on a tissue culture insert following exposure to a potential irritant. Several different types of tissue culture inserts with different membrane growth surfaces, pore sizes and pore densities have been employed in the past. Problems with chemical binding and other incompatibilities with insert membranes can affect the sensitivity of MDCK cells. Cationic surfactants in particular appear to interfere with the passage of fluorescein, leading to underestimation of damage to MDCK cellular function. A blind comparison between MDCK cells cultured on Anopore() and the Millicell-HA inserts using the EU/Home Office set of surfactants indicate that the sensitivity of cells cultured on Anopore inserts appeared to correlate more closely with in vivo data. The ability to microscopically evaluate the status of MDCK cultures through Anopore further recommends this membrane as possibly the better choice for the FL assay.