Biomedical Microdevices

, 21:99 | Cite as

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior

  • Kevin H. Lam
  • Pouriska B. Kivanany
  • Kyle Grose
  • Nihan Yonet-Tanyeri
  • Nesreen Alsmadi
  • Victor D. Varner
  • W. Matthew Petroll
  • David W. SchmidtkeEmail author


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.


Collagen Fibrils Keratocytes Microfluidic PDMS Aligned 



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.

Supplementary material

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  1. C. Chaubaroux, F. Perrin-Schmitt, B. Senger, L. Vidal, J.-C. Voegel, P. Schaaf, Y. Haikel, F. Boulmedais, P. Lavalle, J. Hemmerlé, Tissue Eng. Part C Methods 21, 881 (2015)Google Scholar
  2. P.K. Chaudhuri, C.Q. Pan, B.C. Low, C.T. Lim, Sci. Rep. 6, 19672 (2016)Google Scholar
  3. X. Cheng, U.A. Gurkan, C.J. Dehen, M.P. Tate, H.W. Hillhouse, G.J. Simpson, O. Akkus, Biomaterials 29, 3278 (2008)Google Scholar
  4. P.A. Coghill, E.K. Kesselhuth, E.A. Shimp, D.B. Khismatullin, D.W. Schmidtke, Biomed. Microdevices 15, 183 (2013)Google Scholar
  5. M.J. Dalby, M.O. Riehle, S.J. Yarwood, C.D.W. Wilkinson, A.S.G. Curtis, Exp. Cell Res. 284, 272 (2003)Google Scholar
  6. J.M. Dang, K.W. Leong, Adv. Mater. 19, 2775 (2007)Google Scholar
  7. B.A. David, P. Kubes, Immunol. Rev. 289, 9 (2019)Google Scholar
  8. N. Gjorevski, A.S. Piotrowski, V.D. Varner, C.M. Nelson, Sci. Rep. 5, 11458 (2015)Google Scholar
  9. R. Gruschwitz, J. Friedrichs, M. Valtink, C.M. Franz, D.J. Muller, R.H.W. Funk, K. Engelmann, Invest. Ophthalmol. Vis. Sci. 51, 6303 (2010)Google Scholar
  10. M.D. Guillemette, B. Cui, E. Roy, R. Gauvin, C.J. Giasson, M.B. Esch, P. Carrier, A. Deschambeault, M. Dumoulin, M. Toner, L. Germain, T. Veres, F.A. Auger, Integr. Biol. 1, 196 (2009)Google Scholar
  11. X. Guo, A.E.K. Hutcheon, S.A. Melotti, J.D. Zieske, V. Trinkaus-Randall, J.W. Ruberti, Investig. Ophthalmol. Vis. Sci. 48, 4050 (2007)Google Scholar
  12. J.V. Jester, W.M. Petroll, P.A. Barry, H.D. Cavanagh, Invest. Ophthalmol. Vis. Sci. 36(809) (1995)Google Scholar
  13. D. Karamichos, N. Lakshman, W.M. Petroll, Cell Motil. Cytoskeleton 66, 1 (2009)Google Scholar
  14. D. Karamichos, M.L. Funderburgh, A.E.K. Hutcheon, J.D. Zieske, Y. Du, J. Wu, J.L. Funderburgh, PLoS One 9, e86260 (2014)Google Scholar
  15. W. J. Karlon, J. W. Covell, A. D. Mcculloch, J. J. Hunter, and J. H. Omens, 625, 612 (1998)Google Scholar
  16. N.W. Karuri, S. Liliensiek, A.I. Teixeira, G. Abrams, S. Campbell, P.F. Nealey, C.J. Murphy, J. Cell Sci. 117, 3153 (2004)Google Scholar
  17. J. D. Kiang, J. H. Wen, J. C. Del Álamo, and A. J. Engler, J. Biomed. Mater. Res. - Part A 101 A, 2313 (2013)Google Scholar
  18. P.B. Kivanany, K.C. Grose, W.M. Petroll, Exp. Eye Res. 153, 56 (2016)Google Scholar
  19. P. Kivanany, K. Grose, N. Yonet-Tanyeri, S. Manohar, Y. Sunkara, K. Lam, D. Schmidtke, V. Varner, W. Petroll, J. Funct. Biomater. 9, 54 (2018)Google Scholar
  20. L.B. Koh, I. Rodriguez, S.S. Venkatraman, Biomaterials 31, 1533 (2010)Google Scholar
  21. S. Koo, R. Muhammad, G.S.L. Peh, J.S. Mehta, E.K.F. Yim, Acta Biomater. 10, 1975 (2014)Google Scholar
  22. S. Köster, J.B. Leach, B. Struth, T. Pfohl, J.Y. Wong, Langmuir 23, 357 (2007)Google Scholar
  23. B. Lanfer, U. Freudenberg, R. Zimmermann, D. Stamov, V. Körber, C. Werner, Biomaterials 29, 3888 (2008)Google Scholar
  24. B. Lanfer, F.P. Seib, U. Freudenberg, D. Stamov, T. Bley, M. Bornhäuser, C. Werner, Biomaterials 30, 5950 (2009)Google Scholar
  25. L. Lara Rodriguez, I.C. Schneider, Integr. Biol. 5, 1306 (2013)Google Scholar
  26. P. Lee, R. Lin, J. Moon, L.P. Lee, Biomed. Microdevices 8, 35 (2006)Google Scholar
  27. H.Y. Lou, W. Zhao, Y. Zeng, B. Cui, Acc. Chem. Res. 51, 1046 (2018)Google Scholar
  28. K. Metavarayuth, P. Sitasuwan, X. Zhao, Y. Lin, Q. Wang, ACS Biomater. Sci. Eng. 2, 142 (2016)Google Scholar
  29. M. Miron-Mendoza, E. Graham, S. Manohar, W.M. Petroll, Matrix Biol. 64, 69 (2017)Google Scholar
  30. R. Muhammad, G.S.L. Peh, K. Adnan, J.B.K. Law, J.S. Mehta, E.K.F. Yim, Acta Biomater. 19, 138 (2015)Google Scholar
  31. L. Muthusubramaniam, L. Peng, T. Zaitseva, M. Paukshto, G.R. Martin, T.A. Desai, J Biomed Mater Res Part A 100A, 613 (2012)Google Scholar
  32. K. E. Myrna, R. Mendonsa, P. Russell, S. A. Pot, S. J. Liliensiek, J. V Jester, P. F. Nealey, D. Brown, and C. J. Murphy, Invest Ophthalmol Vis Sci 53, 811 (2012)Google Scholar
  33. W.M. Petroll, P.B. Kivanany, D. Hagenasr, E.K. Graham, Investig. Ophthalmol. Vis. Sci. 56, 7352 (2015)Google Scholar
  34. D. Phu, L.S. Wray, R.V. Warren, R.C. Haskell, E.J. Orwin, Tissue Eng Part A 17, 799 (2011)Google Scholar
  35. E.T. Roussos, J.S. Condeelis, A. Patsialou, Nat. Rev. Cancer 11, 573 (2011)Google Scholar
  36. N. Saeidi, E.A. Sander, J.W. Ruberti, Biomaterials 30, 6581 (2009)Google Scholar
  37. N. Saeidi, E.A. Sander, R. Zareian, J.W. Ruberti, Acta Biomater. 7, 2437 (2011)Google Scholar
  38. N. Saeidi, K.P. Karmelek, J.A. Paten, R. Zareian, E. DiMasi, J.W. Ruberti, Biomaterials 33, 7366 (2012)Google Scholar
  39. A. Saez, M. Ghibaudo, A. Buguin, P. Silberzan, B. Ladoux, Proc. Natl. Acad. Sci. 104, 8281 (2007)Google Scholar
  40. K.E. Sung, G. Su, C. Pehlke, S.M. Trier, K.W. Eliceiri, P.J. Keely, A. Friedl, D.J. Beebe, Biomaterials 30, 4833 (2009)Google Scholar
  41. T. Tabata, Development 131, 703 (2004)Google Scholar
  42. A.I. Teixeira, G.A. Abrams, P.J. Bertics, C.J. Murphy, P.F. Nealey, J. Cell Sci. 116, 1881 (2003)Google Scholar
  43. A. I. Teixeira, P. F. Nealey, and C. J. Murphy, J. Biomed. Mater. Res. - Part A 71, 369 (2004)Google Scholar
  44. J. Torbet, M. Malbouyres, N. Builles, V. Justin, M. Roulet, O. Damour, Å. Oldberg, F. Ruggiero, D.J.S. Hulmes, Biomaterials 28, 4268 (2007)Google Scholar
  45. J.R. Tse, A.J. Engler, PLoS One 6, e15978 (2011)Google Scholar
  46. E. Vrana, N. Builles, M. Hindie, O. Damour, A. Aydinli, V. Hasirci, J Biomed Mater Res Part A 84A, 454 (2008)Google Scholar
  47. B.M. Whited, M.N. Rylander, Biotechnol. Bioeng. 111, 184 (2014)Google Scholar
  48. S. Zhong, W. E. Teo, X. Zhu, R. W. Beuerman, S. Ramakrishna, and L. Y. L. Yung, J. Biomed. Mater. Res. - Part A 79, 456 (2006)Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BioengineeringUniversity of Texas at DallasRichardsonUSA
  2. 2.Department of OphthalmologyUniversity of Texas Southwestern Medical Center at DallasDallasUSA
  3. 3.Department of SurgeryUniversity of Texas Southwestern Medical Center at DallasDallasUSA

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