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Dipika Patel, Aran Sisley, Jie Zhang, Charles NJ McGhee; Emulating the corneal stroma in vitro: Seeding a novel collagen scaffold with keratocytes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):888. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have developed a collagen-based scaffold that has the potential to be used as a corneal substitute for transplantation. The aim of this study was to investigate seeding the scaffold with keratocytes to emulate the corneal stroma in vitro.
Keratocytes were isolated from cadaveric porcine corneas and cultured in a serum-free sphere-forming culture system for 7-10 days. Keratocyte spheres were injected vertically into collagen scaffolds and cultured in serum-free medium supplemented with IGF-II. Scaffolds and keratocyte migration and morphology were imaged using light microscopy. Scaffold contraction and keratocyte migration were measured with NIS Elements, Photoshop and Leica Application Suite. Keratocyte phenotype was characterised by labelling with antibodies directed against keratocan and alpha smooth muscle actin (αSMA) and imaged with confocal microscopy.
There was no significant difference in contraction of cellularized and cell free scaffolds radially or vertically at each of 12 time points measured across 46 days (p>0.05 for each time point). Keratocytes remained viable in the scaffolds for up to 9 months and migrated radially away from the injection site in sheets, similar to the distribution of keratocytes between collagen lamellae in native cornea. The mean maximum rate of migration was 50±12µm/day (n=10, range 29- 72µm/day). Migrating keratocytes had a polar morphology with multiple processes extending in the direction of migration. After at least 9 weeks post-injection, keratocytes took on a quiescent phenotype, characterized by loss of polar morphology, development of a more rounded shape, and increased intercellular processes. Punctate labelling of keratocan was observed and there was no filamentous labelling of αSMA. A similar pattern of labelling was observed in human corneal stroma.
We have successfully seeded a collagen scaffold with primary keratocytes and confirmed that these cells have a distribution and phenotype similar to that seen in the human cornea. Further refinement of our model, such as increasing speed and extent of keratocyte migration, is required to fully emulate the corneal stroma in vitro.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Scanning electron microscopy image showing the lamellar structured scaffold.
Confocal microscopy image stack of keratocytes in collagen scaffold (depth of cells indicated by coloured Z scale).
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