An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen

Abstract

Background: Corneal stromal cells (keratocytes) are responsible for developing and maintaining normal corneal structure and transparency, and for repairing the tissue after injury. Corneal keratocytes reside between highly aligned collagen lamellae in vivo. In addition to growth factors and other soluble biochemical factors, feedback from the extracellular matrix (ECM) itself has been shown to modulate corneal keratocyte behavior.

Methods: In this study, we fabricate aligned collagen substrates using a microfluidics approach and assess their impact on corneal keratocyte morphology, cytoskeletal organization, and patterning after stimulation with platelet derived growth factor (PDGF) or transforming growth factor beta 1 (TGFβ). We also use time-lapse imaging to visualize the dynamic interactions between cells and fibrillar collagen during wound repopulation following an in vitro freeze injury.

Results: Significant co-alignment between keratocytes and aligned collagen fibrils was detected, and the degree of cell/ECM co-alignment further increased in the presence of PDGF or TGFβ. Freeze injury produced an area of cell death without disrupting the collagen. High magnification, time-lapse differential interference contrast (DIC) imaging allowed cell movement and subcellular interactions with the underlying collagen fibrils to be directly visualized.

Conclusions: With continued development, this experimental model could be an important tool for accessing how the integration of multiple biophysical and biochemical signals regulate corneal keratocyte differentiation.

Keywords: collagen fibrils; corneal keratocytes; corneal stroma; engineered substrates; extracellular matrix; growth factors; microfluidics; topography; wound healing.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1

Methods and outcomes for fabrication of collagen. (A) Schematic of the microfluidics approach employed for producing aligned fibrillar collagen; (B) Differential interference contrast (DIC) images of random and aligned collagen substrates. Direction of flow during aligned collagen deposition was horizontal (0°); (C) Quantitative analysis of alignment of random and aligned collagen fibrils. Random fibrils showed no preferential alignment, whereas a peak in alignment near 0° was observed for the aligned collagen substrate.

Figure 2

Scanning electron microscopy (SEM) images of collagen substrates. (A) Aligned collagen fibrils on a substrate without a freeze injury; (B) Aligned collagen fibrils and corneal keratocytes (arrowheads) outside the region of the freeze injury, after four days of culture in media containing PDGF; (C) Aligned collagen fibrils and a cell (arrowhead) within the region of the freeze injury, after four days of culture in media containing PDGF; (D) Collagen fibrils in an unaligned substrate. Arrows indicate direction of flow used during collagen deposition in aligned substrates.

Figure 3

Influence of substrate topography on corneal keratocyte differentiation and alignment. (A–I) F-actin labeling of keratocytes plated on aligned fibrillar collagen, random fibrillar collagen, or unpolymerized collagen-coated substrates, four days after incubation in serum-free media, or serum-free media supplemented with PDGF or TGFβ. Collagen is patterned vertically on the aligned substrates; (J) Positive orientation index (OI) values were found for cells on aligned substrates under all three culture conditions (*, p < 0.001), indicating co-alignment of cells with the direction of the aligned collagen fibrils (left panel). However, cells cultured in the presence of PDGF or TGFβ had higher orientation indices on aligned substrates than cells cultured in basal serum free media (One way analysis of variance using images from at 4 independent experiments per condition). Cells cultured on collagen-coated dishes had mean OI values close to zero, indicating random cell alignment (right panel).

Figure 4

Cell patterning during wound repopulation following freeze injury on aligned collagen substrates. Each panel is a montage of F-actin images collected four days after injury. “W” marks the center of the original wound area, and red lines mark the approximate location of the original wound edge. The aligned collagen fibrils are patterned vertically.

Figure 5

Live cell DIC imaging of cell-collagen interactions. Still images from a time-lapse recording of a cell migrating on an aligned collagen substrate under serum free culture conditions, at the leading edge of a freeze injury (wounded area is to the right). The collagen fibrils are aligned horizontally. Membrane ruffling was observed at the leading edge of the cell (A, arrowhead), and the main direction of movement was in parallel with the aligned collagen (compare cell position with respect to black reference arrows in A,B). Interestingly, several thin processes extended from the cell body both parallel and perpendicular (white arrows) to the collagen fibrils, and some of these process persisted over time. Time is relative to when the freeze injury was made. See also Video S1.

Figure 6

Live cell DIC imaging of cell-collagen interactions. Still images from a time-lapse recording of cells migrating on an aligned collagen substrate under serum free + PGDF culture conditions, at the leading edge of a freeze injury (wounded area is to the left). The collagen fibrils are aligned horizontally. (A,B) The leading edge of two cells is observed (black arrows). The leading edges ruffle as the cells extend, and the cells migrate parallel to the alignment of the collagen; (C) Over time, the two cells interconnect and move together at the leading edge. As the cells continue to elongate, the processes became much thinner. Time is relative to when the freeze injury was made. See also Video S2.

Figure 7

Live cell DIC imaging of cell-collagen interactions. Still images from a time-lapse recording of cell migrating on an aligned collagen substrate under serum free + TGFβ culture conditions, near the leading edge of a freeze injury (wounded area is to the right). The collagen fibrils are aligned horizontally. (A) Note that the cell has a circular morphology with processes extending in all directions. Broader processes are often observed (double arrow); (B) Processes 1 and 2 have extended in parallel with the collagen fibrils, whereas process 3 extended perpendicular to the fibril alignment. Time is relative to when the freeze injury was made. See also Video S4.