Characterization of corneal keratocyte morphology and mechanical activity within 3-D collagen matrices

Abstract

The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent corneal keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit corneal keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1-5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, alpha-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P </= 0.001). Time-lapse imaging of keratocytes revealed occasional extension and retraction of dendritic processes with only transient displacements of collagen fibrils, whereas fibroblasts exerted stronger myosin II-dependent contractile forces (P < 0.01), causing increased compaction and alignment of collagen at the ends of the pseudopodia (P < 0.001). At high cell density, both keratocytes and fibroblasts appeared to form a 3-D network connected via gap junctions. Overall, this experimental model provides a unique platform for quantitative investigation of the morphological, cytoskeletal and contractile behavior of corneal keratocytes (i.e. their mechanical phenotype) in a 3-D microenvironment.

Figure 1

Maximum intensity projections of f-actin organization along the z-axis (A, B) or y-axis (C, D). Cells were plated in 3-D matrices and cultured for 24 hours. A&C: Corneal keratocyte. B&D: Corneal fibroblast. Keratocytes had a stellate morphology with numerous cell processes (A) and a more 3-dimensional structure (C). Fibroblasts typically had a bipolar morphology with thin pseudopodial processes (B). Stress fibers were often observed and cells were oriented nearly parallel to the bottom of the culture dish (D). (E) Quantitative analysis of cell morphology. Both the breadth/length ratio and cell height were larger for corneal keratocytes as compared to corneal fibroblasts. (* P < 0.01, ** P < 0.001)

Figure 2

Color overlays of f-actin (green) and tubulin (red) following 24hrs of 3-D culture in serum-free media. A cortical f-actin labeling pattern was observed, and stress fibers were absent. Dendritic processes contained a core of microtubues (arrowheads) with f-actin concentrated near their tips (arrows). Filopodial extension were also observed (double arrows).

Figure 3

Time-lapse DIC images of a corneal keratocyte (A, C) and fibroblast (B, D). Matrix deformation is indicated by red tracks (crosses mark positions prior to adding cytochalasin D). (B, D) Following culture in serum-containing media, Cytochalasin D induced fibroblast elongation and ECM relaxation. (A, B) In contrast, cytochalasin D had little impact on the matrix surrounding the corneal keratocyte, indicating much smaller cellular forces. (E, F) FEM strain maps generated from matrix displacements. Color legend is in dimensionless units ΔL/L (change in length/initial length). Note the higher strains induced by the fibroblast as compared to the keratocyte.

Figure 4

Maximum intensity projection color overlays of f-actin (green) and collagen fibrils (red) following 24hrs of 3-D culture. (A) Keratocytes had a stellate morphology and numerous cell processes which ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. (B) Increased compaction and alignment of collagen fibrils parallel to the pseudopodial tips was observed at the ends of fibroblasts. Adjacent to the cell body, a more random collagen organization was observed in both cases. (C) Corneal fibroblasts treated with the Rho kinase inhibitor Y-27632 developed a stellate morphology with long dendritic processes, and there was a marked reduction in local collagen matrix reorganization

Figure 5

Local collagen organization. Increased alignment of collagen fibrils at the ends of pseudopodia was observed in fibroblasts as compared to keratocytes or control matrices (no cells), as indicated by higher OI values. Adjacent to the cell body, preferential alignment of collagen was not detected under any condition evaluated. (** P < 0.001)

Figure 6

Collagen fibril density. Significantly greater collagen fibril density was observed surrounding culture of fibroblasts, as compared to culture of keratocytes or control matrices (no cells). Values are % of area occupied by segmented collagen fibrils in individual optical sections. (** P < 0.001, * P < 0.01)

Figure 7

Assessment of global matrix contraction at low cell density. Cell-induced global matrix contraction was significantly greater in fibroblasts, as compared to keratocytes and control matrices (no cells). (** P < 0.001, * P < 0.01)

Figure 8

Cells in 3-D matrices plated at high density and cultured for 24 hours in serum-free media (A, C, E) or 10% FBS (B, D, F) and labeled with phalloidin (green) and/or connexin 43 (red). (A) Keratocytes appeared to form an interconnected network at high cell density (arrows), which was confirmed by connexin labeling (E). (C) Keratocytes were also flatter than in low density cultures, with fewer extensions along the z-axis. For fibroblasts there was an increase in the number of stress fibers as compared to low density cultures (B), and more substantial matrix compaction (D). (F) Connexin 43 was also observed between cells.

Figure 9

Cells in 3-D matrices plated at high density and cultured for 5 days in10% FBS and labeled with phalloidin (green) and α-SM-actin (red). Approximately 10% of cells showed positive α-SM-actin labeling localized stress fibers, suggesting myofibroblast transformation.

Figure 10

Assessment of global matrix contraction at high cell density. Significant global matrix contraction was produced by both corneal keratocytes and fibroblasts as compared to control matrices without cells at 24 hrs (P < 0.001) and 5 days (P < 0.01). However, the amount of global matrix contraction was higher for fibroblasts at both time points (P < 0.001).