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In this study, we developed a novel method of fabricating substrates with micropatterns of Type I aligned collagen fibrils in a 6-well format that allowed for time-lapse imaging of dynamic changes in keratocyte morphology. Culturing keratocytes on aligned collagen fibrils in the presence of platelet-derived growth factor BB (PDGF-BB) allowed us to characterize the dynamics of cell alignment and migration. To investigate the roles of topography and protein composition on the dynamic features of cell spreading, cell protrusions, and cell motility, we cultured keratocytes on either hydrophobic-coated glass, aligned collagen fibrils, or monomeric collagen with or without a fibronectin coating. The presence of a fibronectin coating delayed the formation of cell protrusions during spreading on all of the substrates tested (e.g., Aquasil-coated glass, monomeric collagen, aligned collagen fibrils), while the presence of aligned collagen fibrils resulted in a ~2-fold reduction in the cell spreading area. The experimental platform developed here allows for parallel experiments and real-time imaging and thus providing a valuable new tool to study the dynamic activity and cell\u2013substrate interactions of corneal keratocytes. This approach will allow for systematic screening of the response of keratocytes and other cell types (e.g., tenocytes, cardiomyocytes, cancer cells) that normally are exposed to aligned collagen topographies.<\/jats:p>","DOI":"10.3390\/mi17070783","type":"journal-article","created":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T05:58:04Z","timestamp":1782799084000},"page":"783","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Microwell Platform for Characterizing the Dynamic Response of Corneal Keratocytes to Biochemical and Biophysical Cues"],"prefix":"10.3390","volume":"17","author":[{"given":"Tarik Z.","family":"Shihabeddin","sequence":"first","affiliation":[{"name":"Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2485-6498","authenticated-orcid":false,"given":"Nathaniel S.","family":"Tjahjono","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Divya","family":"Subramanian","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abbas","family":"Rizvi","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miguel","family":"Miron-Mendoza","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victor D.","family":"Varner","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA"},{"name":"Department of Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6404-318X","authenticated-orcid":false,"given":"David W.","family":"Schmidtke","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA"},{"name":"Department of Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1016\/j.ophtha.2020.06.042","article-title":"In Pursuit of the Elimination of Corneal Blindness: Is Establishing Eye Banks and Training Surgeons Enough?","volume":"128","author":"Jeng","year":"2021","journal-title":"Ophthalmology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"107709","DOI":"10.1016\/j.exer.2019.107709","article-title":"Corneal Injury: Clinical and Molecular Aspects","volume":"186","author":"Barrientez","year":"2019","journal-title":"Exp. 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