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AIM/BACKGROUND: To compare the effects of accelerated corneal collagen cross-linking (ACXL) and corneal collagen cross-linking (CXL) on ex vivo-cultured limbal epithelial cells (LECs). METHODS: Day 14 cultured LECs were either unexposed (control) or exposed to different intensities of ultraviolet-A (UV-A) irradiance for different durations (3 mW for 30 min, 9 mW for 10 min, 18 mW for 5 min and 30 mW for 3 min) in the presence and absence of riboflavin. These cells were further processed for quantitative real-time PCR, vital staining, immunofluorescence staining and fluorescence-activated cell sorting (FACS) staining to evaluate the apoptotic status. Statistical analysis was performed using a Student t test. RESULTS: Vital staining showed a significantly higher (p=0.004) dead cell population with 3 mW for 30 min when compared with 30 mW for 3 min exposure (p=0.225). Quantitative PCR results revealed significantly reduced abcg2 and Δnp63 mRNA levels, while FACS analysis showed an increase in ABCG2-Annexin V positive population in cells exposed to 3 mW for 30 mins. Neither reduction of mRNA expression of abcg2 and Δnp63 nor increase in FACS-stained ABCG2-Annexin V positivity was detected in cells exposed to 30 mW for 3 min. Additionally, enhanced caspase activity was detected with fluorochrome inhibitor of caspases staining and mRNA expression of caspase 3 and 9 was upregulated in cells exposed to 3 mW for 30 min, but not at 30 mW for 3 min. CONCLUSIONS: The 30 mW UV-A irradiation used in ACXL appears to be safe on cultured LECs in comparison with 3 mW used in CXL.

Original publication




Journal article


Br J Ophthalmol

Publication Date





272 - 280


Apotosis, Cornea, Ocular surface, Stem Cells, ATP Binding Cassette Transporter, Subfamily G, Member 2, ATP-Binding Cassette Transporters, Annexin A5, Apoptosis, Caspase 3, Caspase 9, Cells, Cultured, Collagen, Corneal Stroma, Cross-Linking Reagents, Epithelial Cells, Flow Cytometry, Fluorescent Antibody Technique, Indirect, Humans, Limbus Corneae, Neoplasm Proteins, Photosensitizing Agents, RNA, Messenger, Real-Time Polymerase Chain Reaction, Riboflavin, Transcription Factors, Tumor Suppressor Proteins, Ultraviolet Rays