Summary: A way to assess the acute and chronic toxic or protective effects of a novel drug candidate or topical formulation.
The corneal epithelium provides an important barrier against fluid loss and protection from the external environment. Disease or injury to the corneal epithelium through viral infection, penetration of chemicals applied topically, or exposure to toxic radiation results in toxicity, irritation, or inflammation. Such reactions in the cornea can cause loss of visual acuity.
|Cells or tissues||Human corneal epithelial cells (HCE-T, RIKEN, Japan)|
|Viability assessment||HCE-T cells are grown on 96-well plates. The cells can be exposed to testing molecules or formulations for a period from a few minutes to 3-4 days. Cytotoxicity is measured using resazurin reduction assay (Nociari et al., 1998). The resazurin assay offers a simple, rapid, and sensitive measurement for the viability of HCE-T cells (Hakkarainen et al., 2016). A cell permeable redox indicator resazurin (7-Hydroxy-3H-phenoxazin-3-one-10-oxide) is used to measure oxidation-reduction reactions in viable cells. The non-fluorescent resazurin is irreversibly reduced by metabolically active cells to highly fluorescent metabolite resorufin (7-Hydroxy-3H-phenoxazin-3-one). Resorufin formation is measured by using Cytation 3 multi-mode reader (BioTek Instruments, Winooski, VT, USA). The quantity of resorufin produced is proportional to the number of viable cells.|
|Positive control||Benzalkonium chloride (BAC)|
|Read-outs||1. Cell viability (%)|
2. IC50 value (50% cell viability) for positive control
3. IC50 value is calculated for study compound if applicable
Outcomes and Read-Outs
- Hakkarainen JJ, Reinisalo M, Ragauskas S, Seppänen A, Kaja S, Kalesnykas G. Acute cytotoxic effects of marketed ophthalmic formulations on human corneal epithelial cells. Int J Pharm. 2016 Sep 10;511(1):73-78. doi: 10.1016/j.ijpharm.2016.06.135.
- Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H. An SV40-immortalized human corneal epithelial cell line and its characterization. IOVS 1995,36:614–621
- Nociari et al. A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Methods. 1998; 213(2):157-167. PMID: 9692848 DOI: 10.1016/s0022-1759(98)00028-3