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Audrey E. Hutcheon, Dimitrios Karamichos, Xiaoqing Q. Guo, James D. Zieske; Corneal Epithelium Stimulates Fibrosis in a 3D Corneal Fibroblast Model. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1853.
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The integrity of the cornea is critical for vision, but healing due to trauma or disease may lead to opacity and vision impairment caused by scarring or fibrosis. The healing process is partly regulated by the epithelial-stromal interactions. In this study we used an in vitro model where the interaction between the epithelium and a self-secreted fibroblast matrix can be examined.
An in vitro 3-dimensional (3D) co-culture model comprised of human corneal epithelial cells (HCEC) and human corneal fibroblasts (HCF) stimulated to secrete a matrix by a stabilized ascorbic acid (sAsc) was used. HCF were stimulated with sAsc and grown for three weeks. At the end of the third week, HCEC were added to the culture. The co-cultures were grown for 4 days and then the HCEC layer was air-lifted for an additional 4 days. Co-cultures were processed for indirect-immunofluorescence microscopy (whole mount and frozen sections) and transmission electron microscopy (TEM). As a control, co-cultures were compared to 4 week HCF cultures without HCEC.
Our data shows that the epithelial cell layer stratified to 3-4 layers, while the self-assembled HCF matrix reached a thickness of 30 microns. High magnification TEM revealed that notable patches of basement membrane were present. Our co-cultures were tested for specific fibrotic markers. Cellular fibronectin (cFN) was expressed subjacent to the epithelial layer and α-smooth muscle actin (SMA) was present in many of the underlying HCF. Little, if any, cFN or SMA were present in the HCF control cultures. We also observed the deposition of type I, III and V collagens.
Application of HCEC resulted in a fibrotic response by the self-assembled HCF matrix, mimicking the wound response observed in mouse and rat after a keratectomy. It also resulted in the beginning formation of a basement membrane; therefore, the data suggests that this co-culture system can be used as a model of human wound repair. This model has great potential for the examination of the HCEC/HCF interactions during regulation of fibrosis and regeneration.
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