Figure 1.
Necessary materials to build a curved cellulose insert for in vitro epithelial surface reconstruction.
a) silicone rings and silicone ring die, b) top and bottom of eye form, c) cellulose insert before forming, d) final curved insert, e) final curved insert with one silicone ring.
Figure 2.
Curved stratified epithelium following exposure to MTT.
Surface coverage by formazan indicates well stratified and viable cells.
Figure 3.
Squamous epithelial cells arranged in layers, firmly attached to the underlying cellulose filters.
H&E, original magnification 63X.
Figure 4.
Effect of BAK and PBS-soaked lens on cellular viability of curved stratified epithelium.
Cells were incubated for 6 and 24 hours with solutions of BAK or PBS-soaked contact lenses. Viability was measured by the MTT assay and is expressed as a percentage relative to cells grown in the absence of a contact lens (control). n = 5, *significantly different from BA PBS control, p<0.05. BA PBS: PBS-soaked balafilcon A lens.
Figure 5.
Effect of ocular surfactants on cellular viability of curved stratified epithelium.
Cells were incubated for 6 and 24 hours with solutions of different poloxamine surfactants at different concentrations. Viability was measured by the MTT assay and is expressed as a percentage relative to cells grown in the absence of a contact lens (control). n = 5, *significantly different from BA PBS control, #significantly different between 6 and 24 hours, p<0.05, T1304: Tetronic 1304, T904: Tetronic 904.
Table 1.
Effect of surfactant, BAK, and PBS-lens exposure on the metabolic activity of corneal epithelial cells in a curved, stratified culture.
Figure 6.
Effect of BAK exposure and PBS-soaked lens on (a) CD49c and (b) CD29 integrin expression.
Following exposure to stimuli after 6 or 24 hours, the curved, stratified cultures of corneal epithelial cells were digested with trypsin and EDTA to form the adherent cell population. CD49c and CD29 expression was measured by flow cytometry and is expressed as a ratio relative to its expression on cells incubated with no lens or BAK (control). n = 3 to 5, *significantly different from PBS-soaked lens, p<0.05. BA PBS: PBS-soaked balafilcon A lens.
Table 2.
Effect of BAK exposure and PBS-soaked lenses on cell integrin expression in a curved, stratified culture of corneal epithelial cells after 6 and 24 hours.
Figure 7.
Effect of BAK exposure and PBS-soaked lens on caspase expression in the (a) adherent and (b) supernatant populations.
Following exposure to stimuli after 6 or 24 hours, the curved, stratified cultures of corneal epithelial cells were digested with trypsin and EDTA. Caspase expression was measured by flow cytometry and is expressed as a ratio relative to its expression on cells incubated with no lens or BAK (control). n = 3 to 5, *significantly different from PBS-soaked lens, p<0.05. BA PBS: PBS-soaked balafilcon A lens.
Figure 8.
Effect of BAK exposure and PBS-soaked lens on PI staining in the (a) adherent and (b) supernatant populations.
Following exposure to stimuli after 6 or 24 hours, the curved, stratified cultures of corneal epithelial cells were digested with Trypsin and EDTA. PI staining was measured by flow cytometry and is expressed as a ratio relative to its expression on cells incubated with no lens or BAK (control). n = 3–5, *significantly different from PBS-soaked lens, p<0.05.
Figure 9.
Cell death following BAK exposure as measured by PI staining.
The histograms display PI staining in the supernatant (a) and adherent (b) cell population following 24 hour exposure to 0.01% BAK. The grey histogram represents cells from the no lens, no solution control; and the black histogram represents cells exposed to BAK. The positive peak for PI (>10 arbitrary fluorescent units) can be observed in both the control and BAK supernatant population, as cells slough off the stratified culture. A significant increase in cells staining positive for positive for PI can be observed following BAK exposure.