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Cell Motility Assays on Tissue Culture Dishes via Non-Invasive Confinement and Release of Cells Girish Kumar,† Jin-Jun Meng,‡ Wallace Ip,‡ Carlos C. Co,† and Chia-Chi Ho*,† Department of Chemical and Materials Engineering, Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, Cincinnati, Ohio 45221 Received February 4, 2005. In Final Form: July 5, 2005 In vitro cell migration assays are useful for screening bioactive agents that regulate angiogenesis, tumor metastasis, would healing, and immune responses by effecting changes in the rate of cell migration. Here we have developed a noninvasive in vitro migration assay that operates through release of confluent groups of cells initially confined within patterns of cell-resistant polyelectrolyte. Cell-resistant patterns of polyelectrolyte, separating groups of confluent cells, are rendered cell adhesive by adsorption of a second, cell adhesive polyelectrolyte of opposite charge; thereby, resulting in migration of cells into the separating regions. By dynamically controlling cell-surface interactions through self-assembly of cell-adhesive and cell resistant polyelectrolytes, this method eliminates the need to mechanically wound cells, as is done in current cell migration assays. The utility of this technique in identifying molecules and mechanisms that regulate cell migration is demonstrated by its application as an assay for the effects of platelet derived growth factors, cytoskeleton disrupting agents, and Merlin overexpression, on the migration of NIH 3T3 fibroblasts.

Introduction Cell migration plays a pivotal role in embryogenesis, tissue morphogenesis, angiogenesis, wound healing, and cancer metastasis. Investigations of mechanisms involved in cell migration,1,2 wound healing,3 and screening agents that influence cell motility4 require quantification of cell migration in vitro. Existing cell motility assays are principally modifications of the Boyden chamber chemotaxis assay,5 wound “healing” assays,6 or observation of single cells in culture.7 In the classic Boyden chamber chemotaxis assay, cells are seeded on one side of a membrane, and the rate of appearance of cells on the other side of the membrane is monitored. Boyden chamber assays are widely used and are commercially available. However, the technique requires cells to be trypsinized and fixed or stained for counting and is further limited to tracking the movement of individual cells. To study the migration of coherent groups or confluent monolayers of cells, representative of processes in vivo, “wound healing” assays have been developed, wherein a confluent monolayer of cells in culture is mechanically, optically, or electrically scratched with a sterile tip, blade, lasers, or other devices. “Wound healing” assays, although conceptually simple and straightforward to implement, are severely complicated by interferences from dead cell debris that block paths for cell movement and * To whom correspondence should be addressed. Phone: (513) 5562438. Fax: (513) 5563473. E-mail: [email protected]. † Department of Chemical and Materials Engineering. ‡ Department of Cell Biology, Neurobiology, and Anatomy. (1) Hauck, C. R.; Hsia, D. A.; Schlaepfer, D. D. J. Biol. Chem. 2000, 275, 41092-41099. (2) James, M. F.; Beauchamp, R. L.; Manchanda, N.; Kazlauskas, A.; Ramesh, V. J. Cell Sci. 2004, 117, 2951-2961. (3) Zhao, M.; Song, B.; Pu, J.; Forrester, J. V.; McCaig, C. D. FASEB J. 2003, 17, 397-406. (4) Sengupta, S.; Xiao, Y. J.; Xu, Y. FASEB J. 2003, 17, U468-U491. (5) Boyden, S. V. J. Exp. Med. 1962, 115, 453-466. (6) Nobes, C. D.; Hall, A. J. Cell Biol. 1999, 144, 1235-1244. (7) Rajah, T. T.; Abidi, S. M. A.; Rambo, D. J.; Dmytryk, J. J.; Pento, J. T. In Vitro Cell. Dev. Biol.-Anim. 1998, 34, 626-628.

molecules released by wounded cells that alter artificially the rate of cell migration.8 Ostuni et al.9 and Folch et al.10 have developed a mechanical technique for depositing cells into poly(dimethylsiloxane) microwells to pattern and release cells. In this approach, cells are confined within the pores of the elastomeric membrane and subsequently released by peeling off the membrane. Although very effective, physical manipulation and removal of the thin membranes require considerable dexterity. Moreover, the technique is limited to cells that can be incubated in serum free media, which is necessary to avoid cell attachment on the membranes. Here we present a straightforward cell migration assay that can be carried on standard tissue culture dishes. By dynamically controlling cell-surface interactions within gaps separating confluent groups of cells, this technique obviates the need to wound confluent monolayers of cells or mechanically confine and release cells. Cell patterning techniques have been used to study and manipulate the behavior of individual cells.11-16 Central to most of these studies is the microcontact printing of cell-adhesive and cell-resistant patterns of self-assembled alkanethiol monolayers pioneered by Whitesides and colleagues for controlling the spatial location, size, and shape of cells.17,18 The operational principle of this approach relies on the preferential attachment of cells (8) Schilling-Schon, A.; Pleyer, U.; Hartmann, C.; Rieck, P. W. Exp. Eye Res. 2000, 71, 583-589. (9) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811-7819. (10) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388-392. (11) Parker, K. K.; Brock, A. L.; Brangwynne, C.; Mannix, R. J.; Wang, N.; Ostuni, E.; Geisse, N. A.; Adams, J. C.; Whitesides, G. M.; Ingber, D. E. FASEB J. 2002, 16 (10), 1195-1204. (12) Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. J. Neurosci. 1988, 8, 4098-4120. (13) Matsuda, T.; Sugawara, T. J. Biomed. Mater. Res. 1995, 29, 749-756. (14) Bhatia, S. N.; Yarmush, M. L.; Toner, M. J. Biomed. Mater. Res. 1997, 34, 189-199. (15) Liu, V. A.; Jastromb, W. E.; Bhatia, S. N. J. Biomed. Mater. Res. 2002, 60, 126-134. (16) Dike, L. E.; Chen, C. S.; Mrksich, M.; Tien, J.; Whitesides, G. M.; Ingber, D. E. In Vitro Cell. Dev. Biol.-Anim. 1999, 35, 441-448.

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within micropatterns of the extracellular matrix and the exclusion of cells from the background region, typically coated with oligoethyleneglycol-terminated thiol. Variations of the microcontact printing approach have since been reported that replace alkanethiols with polymers,13,19 extracellular matrix proteins,10,14,20,21 and cell adhesive peptides22,23 as “inks” to limit the adhesion and spreading of cells within printed micropatterns. We have previously reported the use of copolymers of oligoethyleneglycol methacrylate and methacrylic acid poly(OEGMA-co-MA) for the micropatterning of cells and proteins on chitosan substrates under culture conditions for extended periods of time.24 Here, we demonstrate how poly(OEGMA-co-MA) can be used as part of an in vitro cell migration assay that can be performed on standard tissue culture dishes, without the need to physically wound the cell monolayer. In this assay, cells are first confined on tissue culture dishes patterned with cell-resistant poly(OEGMA-co-MA). Because of the strong acid-base binding of cell-adhesive chitosan to poly(OEGMA-co-MA), the cellresistant patterns of poly(OEGMA-co-MA) can be rendered cell-adhesive in a biocompatible and noninvasive manner, thereby effecting release and migration of cells into the previously cell-resistant regions. Different from the elegant studies reported by Mrksich and co-workers25-27 that require electroactive substrates, the approach described here can be performed on standard tissue culture dishes. Materials and Methods Materials. Tissue culture dishes were purchased from Fisher Scientific (Catalog No. 430166) and used as received. These hydrophobic polystyrene dishes were rendered hydrophilic through plasma treatment by the manufacturer to support cell attachment and spreading.28,29 Poly(dimethylsiloxane) (PDMS; Sylgard 184) was obtained from Dow Corning (Midland, MI). Random copolymers of oligoethyleneglycol methacrylate and methacrylic acid poly(OEGMA-co-MA) with an OEGMA weight ratio of 0.8 were prepared following the procedure described previously.24 Chitosan (182 kDaltons, 69% deacetylation) was a gift from Tri-Corporation (Alpharetta, GA). Texas Red and fluorescein conjugated bovine serum albumin (BSA), Alexa 488phalloidin, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Molecular Probes (Eugene, OR). Phosphate buffer saline was purchased from Fluka (St. Louis, MO). Iscove’s modified Dulbecco’s medium (IMDM) and fetal bovine serum (FBS) were purchased from Cambrex biosciences (Walkersville, (17) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264 (5159), 696-698. (18) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276 (5317), 1425-1428. (19) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. J. Biomed. Mater. Res. 1993, 27, 1243-1251. (20) Hammarback, J. A.; Palm, S. L.; Furcht, L. T.; Letourneau, P. C. J. Neurosci. Res. 1985, 13, 213-220. (21) Miyamoto, S.; Ohashi, A.; Kimura, J.; Tobe, S.; Akaike, T. Sens. Actuator B-Chem. 1993, 13, 196-199. (22) Matsuzawa, M.; Liesi, P.; Knoll, W. J. Neurosci. Methods 1996, 69, 189-196. (23) Patel, N.; Padera, R.; Sanders, G. H. W.; Cannizzaro, S. M.; Davies, M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Shakesheff, K. M. FASEB J. 1998, 12, 1447-1454. (24) Kumar, G.; Wang, Y. C.; Co, C.; Ho, C. C. Langmuir 2003, 19, 10550-10556. (25) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Angew. Chem.-Int. Ed. 2001, 40, 1093. (26) Jiang, X. Y.; Ferrigno, R.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 2366-2367. (27) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992-5996. (28) Ertel, S. I.; Chilkoti, A.; Horbett, T. A.; Ratner, B. D. J. Biomater. Sci.-Polym. Ed. 1991, 3, 163-183. (29) Curtis, A. S. G.; Forrester, J. V.; Clark, P. J. Cell Sci. 1986, 86, 9-24.

Kumar et al. MD). Cytochalasin D, nocodazole, and PDGF-BB were purchased from Sigma (St. Louis, MO). Preparation of Poly(OEGMA-co-MA). Random copolymers of OEGMA and MA (Scientific Polymer Products, NY) were prepared by free radical polymerization of 10 wt % methanolic solutions of the two monomers (80:20 OEGMA to MA mass ratio) at 60 °C. Polymerizations were initiated with 1 wt % (with respect to monomer) of 2,2′-azobis(2-amidinopropane) dihydrochloride (Wako, VA) and allowed to react for 16 h. Similar polymerizations, when carried out in water resulted in precipitation of the copolymer product. Preparation of Patterned Tissue Culture Dishes. Micropatterns consisting of parallel grooves 60 µm wide with ridges of varying widths (10, 20, and 30 µm) and a separate micropattern consisting of 200 µm parallel grooves with 200 µm wide ridges were fabricated on silicon wafers using standard photolithographic techniques. From this silicon master, complementary poly(dimethylsiloxane) (PDMS) replicas were prepared using the soft lithography method developed by Whitesides and co-workers30,31 and used as stamps in subsequent microcontact printing steps to form patterns of poly(OEGMA-co-MA) copolymer directly on cell culture dishes. Patterned dishes were sterilized under UV for 12 h before cells were plated. Spatial control of protein adsorption on poly(OEGMA-co-MA) patterned culture dishes was assessed by incubation with fluorescein-conjugated BSA. Masking of poly(OEGMA-co-MA) printed regions by adsorption of chitosan to render these regions cell-adhesive again was accomplished by immersing the culture dish with media containing 2% water-soluble chitosan. Incubation with Texasred conjugated BSA shows adsorption exclusively over the chitosan-masked poly(OEGMA-co-MA). Plasmid Construction and Cell Transfection. The human cDNA encoding merlin isoform I (GenBank accession number L11353) was a gift from Dr. Nikolai Kley (Bristol Meyer Squibb, Princeton, NJ). Merlin cDNA was cloned into the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA) by using the polymerase chain reaction (PCR). The HA peptide (YPYDVPDYA) was fused in-frame to the amino terminal of merlin I. The construct was sequenced to confirm the fidelity of the PCR amplified regions and in-frame ligation of the cloned regions. The construct was transformed into E. coli strain XL-1 Blue, and bacteria were grown in Luria-Bertani (LB) medium. NIH3T3 mouse fibroblasts (ATCC number CCL92) were cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 µg/mL). Transfection was carried out by using Lipofactamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were used after subsequent selection in G418 (Mediatech, Inc., Herndon, VA) at 300-600 µg/mL for two weeks. Merlin overexpression in cells was monitored by western blotting with either anti-HA or anti-merlin antibodies. Cell Culture. NIH 3T3 fibroblasts and fibroblasts overexpressing Merlin were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% serum. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Sub-confluent monolayers were dissociated with 0.01% trypsin solution, re-suspended in IMDM with 10% serum, and then plated on micropatterned culture dishes. Immunostaining. Cells were fixed with 3.7% paraformaldehyde for 10 min, washed in phosphate buffered saline, and then permeabilized with 0.2% Triton X100 for 5 min. Samples were then rinsed with PBS and incubated with Alexa488phalloidin and DAPI to stain for F-actin and the nuclei. Images of the patterned cells were acquired using a Nikon TE-2000 inverted microscope with Metamorph software (Ver 6.0r4, Universal Imaging, Westchester, PA). Cell Migration Assay. Cells were plated on 200 µm line patterned tissue culture dishes at a density of approximately 10 000 cells/cm2 and allowed to reach confluence within the lines. Patterned tissue culture dishes were then incubated with complete media containing 2% chitosan (182 kDa, 69% deacety(30) Zhao, X. M.; Xia, Y. N.; Whitesides, G. M. J. Mater. Chem. 1997, 7, 1069-1074. (31) Xia, Y. N.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153-184.

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Figure 1. Schematic of approach for patterning and releasing cells with polyelectrolytes. lation, TRI corporation, Alpharetta, GA) for 20 min, after which the cells were restored in complete media containing no chitosan. Phase contrast images at specific sites were recorded at indicated times using a CCD camera (Spot CAM, Diagnostic Instruments Inc.). In some experiments, the complete media was supplemented with cytochalasin D (2.5 ng/mL), nocodazole (10 ng/mL), and PDGF (10 ng/mL) over the entire period of observation.

Results and Discussion A schematic of this cell migration assay is shown in Figure 1. Patterned poly(dimethylsiloxane) PDMS stamps with pre-designed microstructures are inked with poly(OEGMA-co-MA) and stamped directly onto cell culture dishes by microcontact printing. Protein and cell resistant poly(OEGMA-co-MA) are transferred only to regions of the culture dish that contact the stamp. The poly(OEGMAco-MA) printed regions restrict cell attachment within the unprinted regions of tissue culture dish. After the cells have reached confluence within the unprinted regions, chitosan is added to the media to render the poly(OEGMA-co-MA) printed regions cell adhesive. Acid-base binding of chitosan to poly(OEGMA-co-MA) thus allows patterned cells to migrate out from the confining pattern. Figure 2 shows the efficacy of poly(OEGMA-co-MA) in patterning proteins and cells on standard tissue culture dishes in Figure 1 using a PDMS stamp having 60 µm wide plateaus separated by 30 µm grooves. Patterns of poly(OEGMA-co-MA) ink transfer exclusively from the 60 µm plateaus onto the culture dish. Incubation of the patterned culture dishes with fluorescently labeled BSA protein results in exclusive adsorption of BSA within the 30 µm wide uncoated lines separating the 60 µm wide lines of poly(OEGMA-co-MA) (Figure 2A). When NIH 3T3 fibroblasts were seeded on the patterned culture dish, they attached exclusively within regions of the culture dish that have not been coated with poly(OEGMA-co-MA) (Figure 2B). Actin filaments of the patterned cells orient lengthwise along the axis of the line patterns (Figure 2C).

In contrast, cells on unpatterned control regions of the culture dishes exhibit no spatial organization (Figure 2D). To demonstrate how inert poly(OEGMA-co-MA) regions can be switched to support protein adsorption and thus cell attachment, we use fluorescence microscopy to visualize the spatially and temporally controlled adsorption of BSA proteins. Poly(OEGMA-co-MA) is first patterned as a series of 60 µm wide parallel lines spaced 30 µm apart. Initial incubation with Texas red conjugated BSA protein solutions results in the preferential attachment of proteins within the 30 µm wide lines of bare culture dish surface (Figure 3A). Immersion of the substrate in media containing 2% chitosan for 20 min renders the 60 µm wide lines of poly(OEGMA-co-MA) adhesive to proteins as demonstrated by the adsorption of fluoresceinconjugated BSA protein (Figure 3, panels A and B). The utility of this method as an in vitro migration assay for screening drugs that promote or inhibit cell migration and identifying proteins that regulate cell migration is demonstrated in Figure 4. Confluent NIH 3T3 fibroblasts confined within 200 µm wide lines separating cell-resistant poly(OEGMA-co-MA) were released and allowed to advance into the previously cell resistant poly(OEGMA-coMA) regions by incubating the cells and substrate in complete media containing 2% chitosan for 20 min. The rate of cell migration was examined by phase contrast imaging at various time intervals. Cells in complete media advance in to the 200 µm gap and established a confluent cell layer after 42 h (Figures 4 and 5). With the promigratory agent, platelet derived growth factor (PDGF) (10 ng/mL), added to the complete media, cell migration was enhanced and reached confluence within the 200 µm gap in 36 h. During this time, cell proliferation also proceeded, but was similar in both cases. When compared to wild-type NIH 3T3 fibroblasts, the rate of migration of fibroblasts overexpressing Merlin is much slower, requiring ∼72 h to reach confluence in the 200 µm gap. Note that without chitosan adsorption, cells in complete medium

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Figure 3. Patterning of two different proteins on standard culture dishes. (A) BSA (Texas red conjugated) selectively adsorbs on 30 µm wide lines of the bare culture dish surface separated by 60 µm wide lines of poly(OEGMA-co-MA) (B) Adsorption of chitosan on poly(OEGMA-co-MA) renders these 60 µm lines adhesive to a second protein, BSA (green, fluorescein conjugated), allowing for the sequential complementary patterning of two different proteins. Magnification: 200×.

Figure 2. Proteins and cells patterned on cell culture dishes. (A) Selective adsorption of fluorescently labeled BSA on micropatterns. 60 µm wide lines of poly(OEGMA-co-MA) are separated by 30 µm wide lines of the bare culture dish surface to which proteins adsorb. (B) Phase contrast micrograph of patterned NIH 3T3 fibroblasts. (C) Actin filaments align parallel to the axis of the line patterns within 36 h of seeding. (D) NIH 3T3 fibroblasts in unpatterned control regions of the culture dish. Actin microfilaments (green) were visualized with Alexa488-labeled phalloidin. Cell nuclei were visualized with DAPI (blue). Magnification: 200×.

do remain confined within the micropatterns for at least 96 h (Figure 6). PDGF binds to the platelet derived growth factor receptor (PDGFR) and influences cell growth, chemotaxis, actin reorganization, and anti apoptosis.32 PDGF induced actin reorganization and cell migration are known to be mediated through phosphoinositide 3 kinase(PI3k),33 (32) Heldin, C. H.; Ostman, A.; Ronnstrand, L. Biochim. Biophys. Acta-Rev. Cancer 1998, 1378, F79-F113.

phospolipase Cγ,34,35 protein kinase C,36 and small G proteins of the Rho family.32 Our results demonstrate that the addition of PDGF significantly increased the rate of cell migration. NIH 3T3 fibroblasts, transfected with constructs overexpressing Merlin, had significantly reduced rates of cell migration. Merlin is the product of the neurofibromatosis type 2 (NF2) gene. In humans, loss of Merlin is associated with the development of a variety of tumors including schwannomas, meningiomas, ependymomas, gliomas, and mesotheliomas. Previous studies have shown that Merlin interacts indirectly with the cytoskeleton37 and bears significant homology with the ezrin-radixin-moesin (ERM) proteins.38 The slower rate of migration for Merlin overexpressing fibroblasts is likely due not only to their intrinsically reduced mobility but also partly caused by their reduce rate of cell division. Cells treated with cytochalasin D (2.5 ng/mL) and nocodazole (1 ng/mL), which inhibit actin filaments polymerization and depolymerize microtubules39 respectively, exhibited no observable migration (Figure 7, panels A and B). (33) Fruman, D. A.; Meyers, R. E.; Cantley, L. C. Annu. Rev. Biochem. 1998, 67, 481-507. (34) Kamat, A.; Carpenter, G. Cytokine Growth Factor Rev. 1997, 8, 109-117. (35) Kanazawa, H.; Ohsawa, K.; Sasaki, Y.; Kohsaka, S.; Imai, Y. J. Biol. Chem. 2002, 277, 20026-20032. (36) Derman, M. P.; Toker, A.; Hartwig, J. H.; Spokes, K.; Falck, J. R.; Chen, C. S.; Cantley, L. C.; Cantley, L. G. J. Biol. Chem. 1997, 272, 6465-6470. (37) Scoles, D. R.; Huynh, D. P.; Morcos, P. A.; Coulsell, E. R.; Robinson, N. G. G.; Tamanoi, F.; Pulst, S. M. Nat. Genet. 1998, 18, 354-359. (38) Bretscher, A.; Edwards, K.; Fehon, R. G. Nat. Rev. Mol. Cell Biol. 2002, 3, 586-599. (39) Lee, J. C.; Field, D. J.; Lee, L. L. Y. Biochemistry 1980, 19, 62096215.

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Figure 4. Cell migration assay based on noninvasive release of patterned cells from confinement. NIH 3T3 fibroblasts released from pattern confinement following adsorption of cell-adhesive chitosan onto the poly(OEGMA-co-MA) gap. Cells treated with PDGF (10 ng/mL) migrate faster and reach confluence within in 36 h. Cells overexpressing Merlin migrate slower, requiring 72 h to reach confluence. Magnification: 200×.

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Figure 5. Cells treated with PDGF (10 ng/mL) exhibit increased rate of cell migration. Open symbols show average fractional coverage versus time within the 200 µm wide gap for cells released from pattern confinement. NIH 3T3 cells overexpressing Merlin showed reduced rate of cell migration. Solid circles are results from a conventional wound healing assay wherein the gap is formed by scratching with a pipet tip. Error bars represent one standard deviation from the mean.

Figure 6. NIH 3T3 fibroblasts patterned over culture dish remained confined for at least 96 h when no chitosan is added to the media. Magnification: 200×.

To compare the rate of cell migration obtained using the in vitro cell migration assay developed in this study with the traditional wound healing assays,40 we used a pipet tip to scratch a ∼300 µm gap on a confluent monolayer of NIH3T3 fibroblasts on the tissue culture dishes and measured the rate of cell migration into the gap (Figure 8). The cells covered the gap within 24 h, which is much faster than that for patterned cells released (40) FaberElman, A.; Solomon, A.; Abraham, J. A.; Marikovsky, M.; Schwartz, M. J. Clin. Invest. 1996, 97, 162-171.

Figure 7. Screening assays for the anti-migratory drugs cytochalasin D (2.5 ng/mL, Figure 4, panels A and B), and nocodazole (1 ng/mL, Figure 4, panels C and D). Cells do not migrate even after adsorption of chitosan onto the gap of poly(OEGMA-co-MA). Magnification: 200×.

from confinement, despite the higher cell density used on the patterned substrates. The increased rate of cell migration is likely due to intracellular factors released by wounded cells, as reported by Schilling-Schon et al.8 for corneal endothelial cells. In addition, the wounded gap was originally occupied by cells that would have deposited ECM over the gap, whereas the 200 µm gap in our approach was covered by chitosan. Conclusion We have reported here a new in vitro cell migration assay based on real-time dynamic control of cell-substrate interactions, without the need to wound monolayers of

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Figure 8. Example of cell migration on tissue culture dishes measured using the conventional wound healing assay. The line gap was created by scratching a confluent monolayer of NIH 3T3 fibroblasts on a tissue culture dish with a pipet tip.

cells and thus eliminating potential artifacts introduced by damaged cells. Gaps separating confluent monolayers of cells are coated with a cell-resistant anionic polyelectrolyte, which adsorbs cell-adhesive chitosan, thus allowing cells to attach and migrate across the gaps upon addition of chitosan to the medium. The chitosan, added to the medium to effect release of the cells, is highly biocompatible, exhibits low toxicity, and has been widely used as scaffolds for tissue engineering applications.41,42 In this report, we have investigated the effects of soluble agents and overexpression of a neurofibromatosis gene on cell migration using this population-based assay. The

generic two-step procedure for patterning and releasing cells presented here offers cell biologists an easily accessible method for probing the mechanisms of cell migration through comparisons of the rate of migration of wild-type and mutant cells on standard tissue culture dishes with various agents that potentially inhibit or promote cell migration. LA050332N (41) Bumgardner, J. D.; Wiser, R.; Gerard, P. D.; Bergin, P.; Chestnutt, B.; Marini, M.; Ramsey, V.; Elder, S. H.; Gilbert, J. A. J. Biomater. Sci.-Polym. Ed. 2003, 14, 423-438. (42) Khor, E.; Lim, L. Y. Biomaterials 2003, 24, 2339-2349.