Simplified ex-vivo drug evaluation in ocular surface cells: Culture on cellulose filters of cells obtained by impression cytology.

Drug development, resource- and time-intensive, extensively employs cell-based assays to assess the efficacy and safety of candidate drugs. The widely used immortalized cell lines, experimentally convenient, have limited predictive value. In contrast, ex-vivo models more faithfully reproduce diseases but are technically challenging to establish. To address this need, we developed a simplified process for ex-vivo cell culture, demonstrating its feasibility in ocular surface cells. Conjunctival cells were harvested by impression cytology and grown on mixed cellulose ester membrane filters (MCFs). Human and rabbit conjunctival cells cultured on MCFs are 100% viable at 24 h, and 43% viable at 72 h. A gene expression study evaluating 84 genes involved in ocular inflammation demonstrated that ex-vivo culturing maintains intact the expression of two thirds of these genes in human cells. That these cells are suitable for the assessment of ocular drugs was demonstrated by studying the effect of phosphosulindac (PS), a small molecule under development for the treatment of dry eye disease, in both human and rabbit conjunctival cells. PS, for example, suppressed the expression of CXCL10, a cytokine participating in the pathogenesis of dry eye disease, in human and in rabbit conjunctival cells cultured ex-vivo by 32% and 70%, respectively. Conjunctival cells cultured ex-vivo can be transfected to evaluate mechanistic questions. We successfully transfected such cells with a plasmid expressing luciferase under the control of an IFN-γ-responsive promoter or its control plasmid. IFN-γ stimulated luciferase expression by 85% in cells with the responsive plasmid but not in controls; PS significantly suppressed this induction by 37% without affecting the control plasmid. These findings demonstrate that human and rabbit conjunctival cells cultured ex-vivo with our method are viable and maintain their biological integrity; respond to biological and pharmacological agents; and are transfectable with informative plasmids. The unique advantage of this method is to potentially accelerate the development of novel drugs for the treatment of ocular surface diseases, and to advance our understanding of ocular surface pathophysiology.