Joyce L Covre1, Priscila C Cristovam1, Renata R Loureiro1, Rossen M Hazarbassanov1,2, Mauro Campos1,3, Élcio H Sato1,2, José Álvaro P Gomes1,2. 1. Ocular Surface Advanced Center (CASO), Department of Ophthalmology and Visual Sciences, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil. 2. Cornea and External Diseases Service, Department of Ophthalmology and Visual Sciences, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil. 3. Refractive Surgery Service, Department of Ophthalmology and Visual Sciences, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil.
Abstract
PURPOSE: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. METHODS: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. RESULTS: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. CONCLUSION: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.
PURPOSE: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. METHODS: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. RESULTS: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. CONCLUSION: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.