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Joyce Covre, Priscila Cristovam, Renata Loureiro, Rossen Hazarbassanov, Mauro Campos, Élcio Sato, Jose Gomes; Evaluation of the riboflavin and Ultraviolet light effect on keratocytes cultivated in vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1622.
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© ARVO (1962-2015); The Authors (2016-present)
Evaluate the riboflavin and ultraviolet light effect on human keratocytes cultivated in vitro.
Keratocytes were obtained from human corneal rims remnants of tissue previously used in corneal transplants at the Department of Ophthalmology of UNIFESP/EPM, and cultured in DMEM/F12 medium with FBS until confluence. The cell cultures were characterized by immunofluorescence with antibodies to K3 (epithelial marker), Thy 1(fibroblast marker), α-SMA (myofibroblast), Lumican and Keratocan (keratocyte markers). The corneal stromal cells were covered with collagen (200µL and 500 µL) and 0.1% of riboflavin and were exposed to ultraviolet light (UV) for 30 minutes. After 24 hours, cytotoxicity was determined by MTT assay and cell viability was quantified by means of dye Hoechst 33342/Propidium Iodide.
All cell cultures reached confluence around 20 days. Immunofluorescence demonstrated positive expression of keratocyte markers (Lumican and Keratocan) and negative expression of epithelial (K3), fibroblast (Thy 1) and myofibroblast (alpha-SMA) markers. After riboflavin and UV light exposure, cultivated cells without collagen layer presented higher cytotoxicity with MTT(one way ANOVA, p<.0001) analysis and lower rates of apoptosis and necrosis.
Keratocytes cultures were successfully obtained and characterized by immunofluorescence to Lumican and Keratocan. Collagen proved protective effects against UV light exposure.
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