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Lakshman N Subbaraman, Emma Dare, Chung Ki Fung, David J McCanna, Lyndon William Jones; Establishment of optimal culture media in human corneal epithelial wound healing models. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4337. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Damage to the human corneal epithelium can potentially result in severe vision loss. Corneal epithelial cell damage should be quickly repaired to prevent infection and adequate wound healing is required for corneal transplants and recovery from LASIK surgery. To study corneal epithelial wound healing, an in vitro scratch model and an in vitro exclusion zone model are often used. The purpose of this study was to establish the optimum media to use as a control solution in wound healing models.
Immortalized human corneal epithelial cells were cultured in different growth media. A scratch wound was made on the epithelial cell monolayers and cell recovery was followed for up to 48 hours by measuring the area of the wound. The effect of normoxic and hypoxic conditions on tight junctional integrity and metabolic activity of cells grown in different growth media were also investigated. Using an exclusion zone model, the degree of cell proliferation into the exclusion zone was determined after seven and nine days of growth in cell culture media.
Wound healing with Dulbecco’s Modified Eagle Medium: Nutrient mixture F-12 (DMEM/F-12) was significantly faster than both the keratinocyte serum-free medium (KSFM) (p<0.05) and EpiLife (p<0.05) 10 hours after wounding using the scratch model and nine days after wounding using the exclusion zone technique (p<0.05). In addition, hypoxic culture significantly delayed wound healing by an average of 32.4%. In the culture media DMEM/F-12, human corneal epithelial cells stained for abundant zona occludens-1 (ZO-1), connexion 43 (Cx43) and had a high metabolic activity indicating significant epithelial barrier formation, gap junction formation and high cell viability.
DMEM/F-12 led to superior wound healing under hypoxic and normoxic conditions and in two different wound healing models. DMEM/F-12 appears to be the optimum wound healing control for corneal wound healing models due to superior metabolic activity, wound healing and formation of a greater number of tight junctional proteins in cells grown in this medium over the other media tested.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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