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Audrey Hutcheon, Dimitrios Karamichos, Xiaoqing Guo, James Zieske; Co-cultures of Human Corneal Epithelium and Self-assembled Keratocyte and Fibroblast Matrix. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5240.
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© ARVO (1962-2015); The Authors (2016-present)
During corneal wound repair the epithelium switches from interacting with a mature matrix to a temporary matrix that is partially assembled by wound-healing fibroblasts. The goal of the current investigation was to develop and examine models that mimic the interaction of epithelium interacting with a keratocyte-assembled matrix versus a fibroblast-assembled matrix.
Stromal cells were isolated from corneal explants by placing tissue in either 1 or 10% FBS in DMEM. After culture and passaging, the 1% (keratocyte-like) or 10% (fibroblast-like) cells were plated in Transwell dishes with stabilized Vitamin C (VitC) ± 0.1ng/ml of TGF-β3 and maintained in either 1 or 10% FBS in DMEM for 3 weeks. Immortalized human corneal-limbal epithelial cells (HCLE) were then placed atop the self-assembled matrix and cultured for 4 days; after which, the cultures were airlifted and maintained for an additional 1 or 2 weeks. The co-cultures were processed and examined by transmission electron microscopy (TEM) and indirect-immunofluorescence (IF) for morphology, extracellular matrix components and fibrotic markers. Antibodies against collagens I, III, and V, thrombospondin-1 (TSP-1), cellular fibronectin (cFN), and smooth muscle actin (SMA) were examined.
Cells isolated with 1% serum but cultured without T3 did not assemble a matrix. As observed by TEM, the morphology of the stromal cells isolated using 1% serum had a dendritic appearance, which is characteristic of keratocytes; whereas, the 10% serum cells were spindle shaped, a fibroblastic characteristic. This distinct difference in cell shape was also noted after IF with SMA, which was found to be present in both the keratocyte-like and fibroblast-like matrices. Collagens I, III, and V localization were similar in all cultures. Most interestingly, cFN and TSP-1 expression was greatly reduced in the keratocyte-like co-cultures compared to the fibroblast-like co-cultures.
We have developed models that allow for the comparison of the interaction of human corneal epithelial cells with keratocyte-like and fibroblast-like self-assembled matrices. The fibroblast-like matrix appears to stimulate wound-healing responses (as indicated by TSP-1 and cFN expression) to a greater extent than the keratocyte-like matrix.
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