April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Stratification of Conjunctival Epithelial Cells Prevents BAC-Induced Toxicity
Author Affiliations & Notes
  • G. M. Yanochko
    Drug Safety R & D, Pfizer Global R & D, San Diego, California
  • M. G. Evans
    Drug Safety R & D, Pfizer Global R & D, San Diego, California
  • S. Khoh-Reiter
    Drug Safety R & D, Pfizer Global R & D, San Diego, California
  • B. A. Jessen
    Drug Safety R & D, Pfizer Global R & D, San Diego, California
  • Footnotes
    Commercial Relationships  G.M. Yanochko, Pfizer, E; M.G. Evans, Pfizer, E; S. Khoh-Reiter, Pfizer, E; B.A. Jessen, Pfizer, E.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5198. doi:
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      G. M. Yanochko, M. G. Evans, S. Khoh-Reiter, B. A. Jessen; Stratification of Conjunctival Epithelial Cells Prevents BAC-Induced Toxicity. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5198.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Benzalkonium chloride (BAC) is a widely used preservative in topical ocular formulations. The purpose of this study was to evaluate and compare the toxicity of BAC-containing pharmacologically active ophthalmic drugs with those lacking BAC on monolayer or stratified conjunctival epithelial cells.

Methods: : Monolayer Chang conjunctival epithelial cells were grown in 96-well plates and treated with saline, Xalatan®, Patanol®, TravatanZ® (without BAC), 0.02% BAC alone, or the ocular irritants 70% methanol or 5% saponin for 10 or 30 minutes at 37C followed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay for cell viability. Stratification was induced by growing Chang cells on Transwell filters for 5-7 days at which time cultures were air-lifted (AL) by removal of the apical medium. AL cultures were grown for an additional two weeks, exposed to test compounds and assayed for cell viability as above. Transepithelial electrical resistance (TEER) was monitored throughout the culture period to assess general cell health and stratification. Stratification was confirmed after fixation in 10% formalin, sectioning and histological examination using H&E and PAS stains.

Results: : TEER increased significantly after air-lifting cultures and reached a maximum after ~3 weeks in culture. Histological examination indicated that AL cultures were stratified with ~10-15 cell layers whereas non-AL cultures did not stratify. BAC-containing formulations caused significant loss of cell viability in monolayer cultures compared to control after 10 and 30 minutes of exposure (~90% cell loss). Stratification resulted in significantly increased cell viability in response to BAC exposure: ocular formulations with and without BAC were equivalent with ~10-20% cell loss after 30 minutes. Histological examination of stratified cultures confirmed the general health of the cultures after BAC exposure.

Conclusions: : These results demonstrate that stratification significantly affects cell viability of Chang conjunctival cells in response to BAC-containing ophthalmic preparations. Stratified cultures are a more physiologically relevant way to study BAC-induced toxicity on the conjunctival epithelium.

Keywords: ocular irritancy/toxicity testing • conjunctiva 

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