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XQ Guo, AE K Hutcheon, JD Zieske; Human Corneal Organotypic Cultures Using Untranformed Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3211.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In previous studies, organotypic culture models for the human cornea have been developed using transformed cells and an artificial collagen matrix. We hypothesize that the more in vivo-like the starting cells are, the more in vivo-like the final model will be. In our current study, we are attempting to create an organotypic culture model using untransformed cells, and to allow corneal fibroblasts to synthesize and assemble their own extracellular matrix (ECM). Methods: All tissues used in these studies were obtained from donor human eyes. Human corneal keratocytes were cultured with a vitamin C-derivative, L-ascorbic acid 2-phosphate, to stimulate stratification of the keratocytes and deposition of ECM components. The keratocyte/fibroblast cultures were allowed to grow for 8 weeks. Primary human epithelial cells were then seeded on top of some of these structures and allowed to stratify for 6, 8, 10, or 12 days. After culture, half of the construct was fixed for light microscopy and half was frozen for immunohistochemistry. Indirect immunofluorescence microscopy was performed with antibodies against collagen types VI and V, Ki67, α-smooth muscle actin, keratins 3 and 12, and laminin. Results: (MATRIX) Light microscopy confirmed that the fibroblasts stratified to 10-12 cell layers and that a matrix was deposited. Collagen types V and VI were found to be present at high levels. No a -smooth muscle actin was present and Ki67 was present only in the deepest layers of fibroblasts. (EPITHELIUM) Light microscopy indicated that the epithelium stratified to 3-5 cell layers. Keratin 3 and 12 were present in the superficial layers. Laminin was present in the basal cell layers and appeared to increase with length of culture. Ki67 was present primarily in the basal cell layer. Conclusions: Human keratocytes cultured in the vitamin C derivative stratify and assemble their own matrix that appears to have in-vivo like qualities. This matrix supports epithelial cell growth and differentiation, and may provide a good scaffold for creating a corneal organotypic culture model.
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