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H.M. Hoffman, J.H. Choi, D.P. Clousing, J.L. Ubels, D.L. Meadows; Pre–Clinical in vitro Testing of an Artificial Tear Formulation With a Novel Preservation System . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5588.
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© ARVO (1962-2015); The Authors (2016-present)
During development of a multi–dose artificial tear (AT) containing polyethylene glycol, propylene glycol, hydroxypropyl guar and a novel self–preserving system, in vitro toxicity studies suggested damage to monolayers of transformed human corneal epithelial (HCE–T) cells, with cell junction disruption and vacuolization. The AT, however, caused no damage in vivo to intact or compromised rabbit corneas, suggesting that HCE–T monolayers do not adequately model the intact corneal epithelium. Microscopy showed that multilayered HCE–T cells grown at an air–interface were not damaged by the AT. The purpose of this study was to determine effects of this novel self–preserving formulation on viability of monolayer HCE–T cells and to confirm lack of toxicity to multilayered cells using functional measurements.
Monolayer HCE–T cells were exposed to AT formulations with the self–preservation system for 10–60 minutes. Cells were then passed and proliferation was measured, or cells were subjected to a live/dead assay and analyzed by fluorescence microscopy and flow cytometry. Multilayered HCE–T cells grown on membranes at an air interface were exposed to AT formulations for 10–60 minutes, followed by measurement of fluorescein permeability and electrical resistance. Controls were untreated cells or cells exposed to an AT without the preservation system.
The monolayer HCE–T cells could be passed at high plating efficiency and proliferated normally after exposure to the AT. In the live/dead assay conducted immediately after treatment, the percentage of live cells was equal to control cells and few dead cells could be detected by microscopy. Exposure to the novel AT formulation caused no changes in permeability or resistance of air–interface, multilayered cultures that could be attributed to effects of the formulation, suggesting no damage to junctional complexes.
Although disruption of cell junctions in monolayer HCE–T cells was observed, the cells recover when returned to culture medium, are viable and proliferate normally. The self–preserving AT does not damage multilayer cultures of HCE–T cells grown at an air interface. This is in agreement with in vivo data and shows that the air interface, multilayered culture is a better model of the corneal epithelium for toxicity tests, probably because the superficial cells of the cultures have tight junctions and low permeability like the superficial cells of the in vivo cornea. These experiments show that in vitro toxicity tests must be designed and chosen carefully to avoid erroneous conclusions.
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