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X. Lin, L. Ma, W. M. Petroll; A 3-D Culture Model for Assessing the Mechanics of Fibroblast Migration Through Fibrin Matrix. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6226.
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Wound healing after corneal injury often involves the migration of activated keratocytes (fibroblasts) from the surrounding stroma into a fibrin clot. The purpose of this study was to develop and test an in vitro culture model for studying the mechanics of corneal fibroblast migration from a stromal tissue equivalent into 3-D fibrin matrix.
Human corneal fibroblasts were seeded within hydrated collagen matrices, which were then compressed via application of an external loading force. A 6 mm diameter button of this cell-seeded matrix was placed on a glass bottom culture dish, and a mixture of DMEM with fibrinogen (3mg/mL) and thrombin (1 - 2 U/mL) was poured on top. After 30-60 minutes of incubation at 37º and 5% CO2 to allow fibrin polymerization, these nested constructs were cultured for 1 to 3 days in media containing 10% fetal bovine serum. Constructs were then fixed and labeled with AlexaFluor 488 phalloidin (for f-actin) and TOTO-3 (for nuclei), and cell-matrix interactions were assessed using a combination of fluorescent and reflected light confocal imaging.
Homogenous, randomly organized strands of fibrin were generally visible by confocal reflection microscopy in most control samples (without cells); however, the structure of the matrix was highly sensitive to the experimental conditions used for polymerization (i.e. temperature, thrombin concentration and timing). Cell migration from the inner collagen matrix into the outer fibrin matrix was observed by 1 day after plating. Fibrin density was increased in areas immediately adjacent to the migrating fibroblasts, presumably due to cell-induced compaction and alignment of fibrils. Intracellular stress fibers were often observed, and fibrin was aligned parallel to stress fibers, when present.
This 3-D culture model allows the remodeling of fibrin matrix which occurs during corneal fibroblast migration to be directly visualized. A unique feature is the use of the compressed collagen matrix, which has a geometry, density and stiffness similar to the native corneal stroma. This model could serve as a unique in vitro platform for investigating how fibroblast-induced matrix degradation and remodeling is regulated by specific cytokines present during corneal wound healing.
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