Cytotoxicity Testing of Multipurpose Contact Lens Solutions Using Monolayer and Stratified Cultures of Human Corneal Epithelial Cells

Abstract

Monolayer cultures are highly sensitive to damage by MPS. In contrast, because stratified human corneal-limbal epithelial cultures are resistant to adverse effects of MPS, it is suggested that models that simulate the stratified structure of the corneal epithelium should be used for in vitro toxicologic testing. Caution should be used when interpreting such studies, because in vitro tests may not be predictive of clinical responses to contact lens products that are known to be safe when used as directed.

Effects of Peroxide-Based Contact Lens–Disinfecting Systems on Human Corneal Epithelial Cells In Vitro

“…As previously reported, 18 HCLE cells grow to a confluent, uniform monolayer of typical epithelial morphology (Fig. 1).…”

“…To quantify the effects of the H 2 O 2 formulations and controls on cell viability, confluent monolayers of HCLE cells were exposed to test formulations for 10, 20, or 60 minutes and were subjected to the live/dead assay with flow cytometry, as described by Lim et al 18 Cell viability was determined using a BD FACSCaliber flow cytometer (BD Biosciences) with windows set to differentiate between live and dead cells. Cells demonstrating a high uptake of calcein AM were counted as alive, and those stained with EthD-1 were counted as dead.…”

“…To test the effects of the H 2 O 2 formulations and controls on the barrier function of the corneal epithelium, a modification of the fluorescein leakage test 19 was performed using the method described by Lim et al 18 Stratified HCLE cell constructs grown in culture inserts for 7 days were exposed to H 2 O 2 formulations for 20 or 60 minutes. Sodium fluorescein at 0.02% in HBSS was placed in the inserts and allowed to permeate through cell constructs for 30 minutes at 37°C into the outer wells, which also contained HBSS.…”

“…Images of frozen sections have shown that stratified constructs grow to be 2 to 5 cell layers thick. 18 Morphology Human corneal-limbal epithelial cells were grown to form confluent monolayers or were stratified in 12-well culture plates. Before exposure, cells were imaged using Zeiss AxioVision software and an AxioCam MRc camera on a Zeiss Axiovert 200 inverted microscope with Hoffman Modulation Contrast Optics (Carl Zeiss, Thornwood, NJ).…”

“…Clear Care Ò was neutralized for 6 hours using the provided neutralization case as per the manufacturer's instructions before use in experiments. Hank's Balanced Salt Solution (HBSS) complete (Gibco, Grand Island, NY) and culture medium were used as negative controls, and 0.3% Triton-X 100 (Sigma, St. Louis, MO) in deionized water, a detergent known to damage cells in culture, 18 was used as a positive control.…”