In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.
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This work was supported in part by grants from the National Institutes of Health (R01 EY013322, R01 EY030190), a Trainee Fellowship from the UT-Southwestern Hamon Center for Regenerative Science and Medicine (CRSM) Trainee to KHL, a Pilot and Feasibility grant from the UT Southwestern O’Brien Kidney Research Core Center, and a grant from Research to Prevent Blindness, Inc. and funds from the Office of Vice President of Research at the University of Texas at Dallas. The authors would like to thank Somdutta Chakraborty for assistance with some of the confocal imaging. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Lam, K.H., Kivanany, P.B., Grose, K. et al. A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior. Biomed Microdevices 21, 99 (2019). https://doi.org/10.1007/s10544-019-0436-3