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Ming-Feng Wu, Tanja Stachon, Jiong Wang, Xuefei Song, Achim Langenbucher, Berthold Seitz, Nora Szentmary; Impact of keratocyte supernatant on epithelial cell migration and proliferation after corneal crosslinking (CXL). Invest. Ophthalmol. Vis. Sci. 2014;55(13):505.
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To evaluate the impact of keratocyte supernatant on migration and proliferation of human corneal epithelial cells (HCEC) following crosslinking (CXL), in vitro.
Primary human keratocytes were cultured in DMEM/F12 with 10% FCS. Thereafter, keratocyte cell cultures underwent UVA illumination using light (375 nm) for 4.10 minutes during exposure to 0.05% and 0.1% riboflavin and 20% dextran containing PBS. After removal of the riboflavin containing medium, cultures were incubated in DMEM with 10% FCS for 5 or 24 hours and the supernatant of the cells was collected. HCECs were cultured in DMEM/F12 with 5% FCS, 0.5% DMSO, 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium. Then the HCEC culture medium was replaced by the keratocyte supernatant. Thereafter, HCEC migration was analyzed using scratch assay and cell proliferation was determined by the ELISA-BrdU Kit.
Using riboflavin or illumination separately, there was no significant difference in HCEC migration or HCEC proliferation between treated and control cells in any of the groups (p>0.13). The riboflavin-UVA keratocyte supernatant did not have a significant impact on the HCEC migration, compared to controls at the examined time-points (p>0.39). Proliferation of HCEC with a riboflavin-UVA keratocyte supernatant was unchanged using 0.05% riboflavin conditioned for 5 or 24 hours (p>0.07) and using 0.1% riboflavin conditioned for 5 hours incubation (p=0.16), compared to controls. However, using 0.1% riboflavin concentration and for 24 hours conditioned keratocyte supernatant, proliferation of HCEC was significantly decreased (p=0.03), compared to the control.
Keratocyte secretion does not seem to have an impact on epithelial cell migration in the short term. However, decreases proliferation of HCECs after standard crosslinking therapy after 24 hours, in vitro.
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