With the development of defined media for general and specific use with cell culture, and concern over human cells and prion infections associated potentially with growth factor extracts such as Bovine Pituitary Extract, an animal product free medium has become available. The keratinocyte defined medium employs the same basic medium but with a choice of animal-product containing HKGS, or animal product free supplements (HKGS-V2). Human corneal epithelia cell lines were cultured in these two media and compared for their growth rates, capacity to form tight barriers and for calcium regulation of location of Zonula Occludins 1 (ZO-1) to the cell membrane. The growth rates were no different in the two media, as long as the recommended coating was applied to the culture flask for the HKGS-V2 containing medium. The barrier function was equally tight for the confluent cultures seeded at the same densities. The calcium concentration at 100µM or above resulted in ZO-1 localisation to the cell membrane in either medium. Hence the media are comparable, when the coating is employed. Further experiments are being conducted to establish comparability of response to chronic surfactant treatment.