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C. Alonso-Ron, A. Aracil, J. Gallar, J. Merayo-Lloves, M. Chacon, C. Belmonte; Effect of Neuropeptides in a Human Corneal Epithelium Wound Healing Model in vitro. Invest. Ophthalmol. Vis. Sci. 2010;51(13):376.
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© ARVO (1962-2015); The Authors (2016-present)
To develop an experimental model of human corneal epithelium cells in culture to quantify the healing rate of epithelial corneal wounds of controlled size, under basal conditions and after exposure to different trophic factors.
SV40 immortalized human corneal epithelial cells (HCE; Araki-Sasaki K. et al. , 1995) were grown in 24-wells plates in DMEM:F-12 medium supplemented with 7.5% FBS, 10 ng/mL EGF, 0.5 µg/mL insulin, 1% DMSO and penicillin/streptomycin mixture (100 units-µg/mL) [Complete Feeding Medium, CFM] until confluence. In vitro wounds were produced using a pipette tip centered in each well by a cylindrical piece of methacrylate with a hole in the middle. The wounds were photographed every 12 h and their area was measured using the Image J software (NIH). Estimated rate of healing (ERH) was determined by the slope obtained after fitting the decrease of wound area time course to a line function. The effects of substance P (SP) and Calcitonin Gene Related Peptide (CGRP) at different concentrations were tested in CFM, in DMEM:F-12 supplemented with 0.75% FBS, 1 ng/mL EGF, 0.05 µg/mL insulin, 0.1% DMSO and penicillin/streptomycin (Low Complete Feeding Medium, LCFM), or in DMEM:F-12 supplemented with only penicillin/streptomycin (Basic Medium, BM).
When cells in culture reached confluence they formed a monolayer or a pseudo-monolayer with a morphological appearance ressembling the corneal epithelium. The content in growth factors of the culture medium influenced wound healing rate. No healing occurred in BM, while it was maximal in CFM (ERH = -3.95 ± 0.09) and intermediate in LCFM (ERH = -1.14 ± 0.15). Addition to the culture medium (LCFM) of the SP analog MetOH(11)SP (5-20µM) every 12 h significantly increased ERH. In the same conditions, CGRP did not modify significantly the ERH.
The in vitro model of human epithelium wound healing is very sensitive to external growth factors and may be useful to evaluate the effect of different growth factors on human epithelium cell migration and proliferation following injury.
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