Characterization of Corneal Keratocyte Morphology and Mechanical Activity Within 3-D Collagen Matrices

Abstract

Purpose:: Quiescent corneal keratocytes develop a characteristic morphology and cytoskeletal organization in 2-D culture that is distinctly different than activated corneal fibroblasts. The purpose of this study was to assess corneal keratocyte morphology and mechanical activity in a 3-D culture model.

Methods:: Primary cultures of rabbit corneal keratocytes were plated at low (2 x 10⁴ cells/ml) or high (6 x 10⁵ cells/ml) density inside type I collagen matrices under serum-free conditions, and allowed to spread for 24-48 hours. In some experiments, cells were fixed and stained using AlexaFluor 488 phalloidin, and fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, cells were labeled with calcein AM, and time-lapse (4-D) confocal imaging was performed.

Results:: Corneal keratocytes within collagen matrcies developed a stellate morphology with numerous dendritic cell processes extending 3-dimensionally. The breadth/length ratio for corneal keratoctyes was signficantly higher that previously established for corneal fibroblasts using the same model (0.67 + 0.12 vs. 0.27 + 0.10, p < 0.01), confirming a less bipolar morphology. Dendritic cell processes had numerous branches, and ran a tortuous path both between and along collagen fibrils without any apparent impact on their alignment. A cortical f-actin organization was observed, with increased labeling often observed at the tip of cell processes. At high density, keratocytes were generally flatter, and formed an interconnected 3-D network. Time lapse imaging revealed occasional extension or retraction of dendritic cell processes without significant matrix deformation.

Conclusions:: In contrast to corneal fibroblasts, which develop a bipolar morphology and generate contractile forces that compact and align collagen fibrils, corneal keratocytes assume a stellate morphology and do not generate significant forces on the matrix in 3-D culture. At high density, corneal keratoctyes form an interconnected network within collagen matrices which resembles their organization in vivo. Overall, this experimental model should provides a unique platform for simultaneous investigation of the morphological, cytoskeletal and mechanical behavior of these cells.

Keywords: cornea: stroma and keratocytes • wound healing • imaging/image analysis: non-clinical