Cell Migration and Polarity on Microfabricated Gradients of Extracellular Matrix Proteins Rico C. Gunawan, ² Jonathan Silvestre, ², | H. Rex Gaskins, ‡,§ Paul J. A. Kenis, ²,§ and Deborah E. Leckband* ,²,§ Department of Chemical and Biomolecular Engineering, Department of Animal Sciences, and Institute for Genomic Biology, UniVersity of Illinois at Urbana-Champaign, 600 South Matthews AVenue, Urbana, Illinois 61801 ReceiVed NoVember 21, 2005. In Final Form: February 14, 2006 This paper explores the effects of the surface density and concentration profiles of extra cellular matrix proteins on the migration of rat intestinal IEC-6 cells. Microfluidic devices were used to create linear, immobilized gradients of laminin. This study investigated both the impact of the steepness and local concentrations on the directedness of cell migration. The bulk concentrations of proteins in the feed streams in the mixing device determined the gradient profile and the local concentration of laminin in the device. Two sets of gradients were used to explore cell migration directedness: (i) gradients with similar change in local concentration, i.e., the same gradient steepness, and (ii) different gradients with similar local concentrations. Cells migrated up the gradients, independent of the steepness of the gradients used in this study. At the same local laminin concentration, the migration rate was independent of the gradient steepness. However, cell directedness decreased significantly at high laminin densities. Introduction During embryonic development, cells migrate to form spatially segregated, specialized tissues. This targeted cell migration is required for proper tissue formation. For instance, the formation of the central nervous system depends on directed neurite extension and specific target identification over enormous distances. 1 In addition to morphogenesis, cell migration plays an important role in wound repair, angiogenesis, the inflammatory response, tumor cell metastasis, and tissue engineering. 2 Numerous in vitro studies of cell migration benefited from the use of two-dimensional substrates coated with immobilized proteins and peptides. 3,4 Recent studies showed that axons turn and migrate up an adhesive peptide gradient. 5 Additionally, these substrates were used to show that migration velocity depends on the density of substratum-bound ligand, the concentration of ligand receptor, e.g., integrins, and the receptor-ligand binding affinity. These variables, in turn, affect how cells transmit the intracellular contractile force into a traction force to move forward. In vivo, some cells migrate in response to specific patterns of such stimuli as soluble chemoattractants (chemotaxis) and surface- bound adhesion molecules (haptotaxis). In particular, substrate gradients are thought to direct cell migration in the epithelium of the small intestine 6 (Figure 1A). The intestinal epithelium consists of a monolayer of four different cell types: enterocytes, enteroendocrine cells, Paneth cells, and goblet cells. 7,8 Stem cells proliferate and undergo differentiation into one of the four cell types while migrating from the base of the crypts of Lieberku ¨hn to the base of the villus. 9 The differentiated cells then continue to migrate upward to the tip of the villus where they are finally exfoliated into the intestinal lumen. The rate of epithelial cell migration, which was estimated from isotope tracer studies, ranged from one to two cell positions per hour. 7 The average villus residence time is thus 3-6 days. 8 Extracellular matrix (ECM) proteins beneath the epithelium reportedly influence multiple cellular functions such as prolifera- tion, differentiation, migration, and tissue-specific gene expres- sion. 6 Members of the integrin superfamily primarily mediate these functions. The most significant finding yet to support a possible relationship between ECM protein expression and intestinal cell functions is the spatial gradient of laminin isoforms along the crypt-villus axis (Figure 1A). The expression of laminin-1 gradually increases from the crypt-villus junction to the villus tip. Conversely, laminin-2 expression decreases with * To whom correspondence should be addressed. Phone: 217-244-0793. Fax: 217-333-5052. E-mail: leckband@scs.uiuc.edu. ² Department of Chemical and Biomolecular Engineering. ‡ Department of Animal Sciences. § Institute for Genomic Biology. | These authors contributed equally to this work. (1) Helle, T.; Deiss, S.; Schwarz, U.; Schlosshauer, B. Expt. Cell Res. 2003, 287, 88-97. (2) Lauffenburger, D. A.; Horwitz, A. F. Cell 1996, 84, 359-369. (3) DiMilla, P.; Stone, J.; Quinn, J.; Albelda, S.; Lauffenburger, D. J. Cell Biol. 1993, 122, 729-737. (4) Palecek, S.; Schmidt, C.; Lauffenburger, D.; Horwitz, A. J. Cell Sci. 1996, 109, 941-952. (5) Adams, D. N.; Kao, E. Y.-C.; Hypolite, C. L.; Distefano, M. D.; Hu, W.-S.; Letourneau, P. C. J. Neurobiol. 62, 134-147. (6) Beaulieu, J.-F. Recent Work with Migration/Patterns of Expression: Cell- Matrix Interactions in Human Intestinal Cell Differentiation.; Kluwer Academic Publishers: Hingham, MA, 1997. (7) Potten, C.; Loeffler, M. DeVelopment 1990, 110, 1001-1020. (8) Potten, C. S. Philos. Trans. R. Soc. London B 1998, 353, 821-830. (9) Beaulieu, J.-F. Front. Biosci. 1999, 4, d310-321. Figure 1. (A) In vivo expression profile of laminin isoforms along the crypt-villus axis of the small intestine: laminin-1 (L1), laminin-5 (L5), and laminin-2 (L2). (B) Microfluidic network design used to recreate ECM protein gradients. 4250 Langmuir 2006, 22, 4250-4258 10.1021/la0531493 CCC: $33.50 © 2006 American Chemical Society Published on Web 04/01/2006