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W. M. Petroll, D. Karamichos, L. Ma, N. Lakshman; A Novel Corneal Keratocyte Culture Model Using Compressed Collagen Matrix. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4042. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a novel 3-D culture model which mimics corneal stromal mechanical properties while supporting keratocyte differentiation, transformation and migration.
Rabbit corneal keratocytes were isolated and cultured in serum-free media. To prepare compressed collagen matrices, a 6 ml solution containing cells and type I rat tail collagen (3 mg/ml) was poured into a 3x2x1 cm well, and allowed to polymerize for 1 hour at 37°C. Following polymerization, the matrix was compacted by using compression with a 130g stainless steel block for 5 minutes at room temperature. This process squeezes media out of the matrix and results in the formation of a flat, cell/collagen sheet (~50 µm thick) with high mechanical stiffness (~1 MPa), similar to native corneal stroma. Following compression, 6mm diameter buttons were punched out of the compressed matrix and incubated in serum-free media for 24 hours to allow cell spreading. Media was then replaced with serum-free media supplemented with either PDGF (50 ng/ml), TGFβ1 (10 ng/ml), TGFβ2 (10 ng/ml) or no growth factor (control). Matrices were incubated an additional 1 - 7 days, labeled with AlexaFluor 488 Phalloidin, and imaged using laser confocal microscopy. To study migration, some buttons were nested within acellular uncompressed outer collagen matrices prior to incubation with either PDGF or control media.
Corneal keratocytes maintained in serum-free media within compressed matrices had a broad, convoluted cell body and numerous thin dendritic processes, which extended in all directions from the cell; stress fibers were not detected. Treatment with PDGF induced elongation of corneal keratocytes and development of a less convoluted cell morphology, without inducing stress fiber formation. In contrast, treatment with TGFβ1 & 2 induced the formation of stress fibers and the loss of thin cell processes, suggesting transformation from a dendritic phenotype to a contractile phenotype. In the nested matrix model, PDGF stimulated an increase in keratocyte migration into the uncompressed outer matrix as compared to controls (both number of cells and distance traveled). Reorganization of collagen fibrils within the outer matrix was observed during cell migration.
Corneal keratocytes are able to differentiate normally and respond to growth factors within compressed collagen matrices, which provide a high stiffness, 3-D environment similar to native stromal tissue. In addition, nesting these matrices provides a unique platform for investigating the migratory response of corneal keratocytes following exposure to specific wound healing cytokines.
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