Abstract
Abstract: :
Purpose: In the human cornea, the collagen fibrils that make up the stroma are found to be in sheets that appear to be perpendicular to each other. It is this formation, the pattern in which the stromal cells are spaced and the lack of blood vessels that allows the cornea to be transparent. We have previously shown that by growing untransformed human corneal fibroblasts with Vitamin C, the fibroblasts form their own matrix. In this current study, we continue to examine these cells to see if the fibroblasts themselves align as they form a sheet and if when they stratify, the next sheet of fibroblasts is aligned perpendicular to the first. Thus, laying down a collagen matrix perpendicular to the first. Methods: Human corneal fibroblasts were cultured directly onto 6–well culture plates and allowed to grow in DMEM/10%FBS +/– 1mM Vitamin C for 1–8 weeks. Cultures of human skin fibroblasts served as controls. The cultures were monitored weekly by phase contrast microscopy. After the appropriate time, the cultures were either fixed for TEM or methacrylate, or frozen in OCT and stained for immunofluorescence. For immunofluorescence, the cultures were rinsed briefly with PBS and then incubated for 1 hour with 2.5 µM Calcein–AM in PBS at 37°C. The cultures were then washed with PBS briefly and incubated with fresh PBS for 30 minutes at 37°C. After which, they were fixed with 4% formaldehyde in PBS for 15 minutes. The cultures were washed 3 times with PBS, blocked with 1% Bovine Serum Albumin (BSA) for 10 minutes at room temperature, and incubated with Rhodamine Phalloidin and TOPRO3 in BSA for 1 hour at room temperature. The stain was then removed and the cultures were washed 2 times in PBS and coversliped with Vectashield mounting media. Results: By 1 week, a single layer of aligned fibroblasts is present. By 2 weeks, the fibroblasts have stratified to form two layers of cells, and by phase microscopy, the second layer appears to be aligned perpendicular to the first. By 3–4 weeks, four layers of cells appear to be present. Some of the layers appear to be perpendicular to each other, but the level of disorganization increases as the number of layers increases. Conclusions: With time, human corneal fibroblasts align themselves, and upon stratification, the alignment of the next layer appears to be perpendicular to the first layer. Thus, perpendicular alignment of human corneal fibroblasts may be intrinsic to the cells. However, the increased disorganization of the cell layers with time suggests that additional signaling cues are required to maintain alignment in multiple layers of cells.
Keywords: extracellular matrix • cornea: basic science • cornea: stroma and keratocytes