Poster V3 - Time-lapse Imaging of Keratocyte Morphology in Response to Topography and ECM Composition

Introduction: Corneal injury results in the loss of normal corneal topography, which consists of aligned collagen fibril sheets layered orthogonal to one another. After injury, corneal keratocytes secrete, among other things, Fibronectin (FN) and collagen monomers into the wound area. Fibronectin is thought to act as a provisional matrix that supports cell migration and myofibroblastic transformation (1). Collagen monomers are used by corneal keratocytes to organize the wound site and eventually re-form the normal aligned collagen fibril topography. We previously reported that exposure of keratocytes to adsorbed fibronectin transiently induces them to become activated as indicated by a change in their morphology and α-SMA expression (2). The extent of keratocyte activation was dependent not only on the length of cell culture (2 vs. 5 days) but also on whether the keratocytes were cultured on aligned collagen fibrils or on a flat rigid substrate such as glass. The duration of this transient activated cellular state is unclear, however, because we only analyzed two time points with static fluorescent imaging. In this study, we developed a novel six-well plate assay that allowed us to perform time-lapse microscopy to further investigate the dynamic shape changes of keratocytes in response to different topographies and extracellular matrix composition.

Materials and

Methods: Six-well plates with aquasil treated glass bottoms were fabricated, sterilized, and coated with either collagen monomers, aligned collagen fibrils, or FN. Collagen monomers and FN were adsorbed by creating the appropriate solutions, adding each solution to its own well, and then washing the wells. Collagen fibrils were created by modifying a procedure previously described (1). Briefly, PDMS stamps with straight channels were fabricated to fit on the glass within the well of a six-well plate, and collagen solution was run through the stamps to form aligned fibrils. Normal rabbit corneal keratocytes were seeded within each well and the plate was set up for a time lapse. Phenotypic changes observed in branching within the videos were quantified using ImageJ to calculate solidity as described by Lobo et al (3) where Solidity = cell area/convex hull area. A solidity value of 1 means a solid object, while lower values represent objects with irregular boundaries or highly branched.

Results, Conclusions, and Discussions:

Results: We observed that cells cultured on collagen monomers and collagen fibrils initially had a spread morphology (indicated by the high solidity value) that began to branch (indicated by the lower solidity value) after about 24 hours. By 60 hours, the cells on both collagen monomers and aligned fibrils returned to a very branched, quiescent morphology. In contrast, cells cultured on FN exhibited a polygonal morphology and remained spread for a longer period (about 40 hours) before beginning to branch. By 60 hours, cells on FN had a branched morphology but the branching was less than that of cells on collagen monomers and aligned collagen fibrils. Cells cultured on glass showed a trend similar to collagen monomers and fibrils, but with higher solidity values although not as high as those seen with cells cultured on FN.

Conclusions: The development of a novel 6-well plate assay allowed us to simultaneously investigate the dynamic effects of several different parameters (e.g. topography, ECM composition) on keratocyte behavior over 5 days. With this assay we were able to characterize dynamic differences in keratocyte morphology in response to fibronectin, collagen, and the topography of aligned collagen fibrils.

References: 1. Kivanany PB, et al. “An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen”, J Functional Biomaterials 9, 54, 2018.
2. Lam KH, et al., “Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin coated Surfaces”, J Funct Biomater. 14:217, 2023.
3. Lobo, J., See, E. Y. S., Biggs, M. & Pandit, A. An insight into morphometric descriptors of cell shape that pertain to regenerative medicine. Journal of Tissue Engineering and Regenerative Medicine vol. 10 539–553 (2016).

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