Cellular and Molecular Bioengineering, Vol. 3, No. 1, March 2010 ( 2010) pp. 4049 DOI: 10.

1007/s12195-010-0108-0

Measuring Traction Forces in Long-Term Cell Cultures

CYNTHIA MANN and DEBORAH LECKBAND
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801, USA
(Received 21 December 2009; accepted 12 February 2010; published online 24 February 2010)

AbstractThis report describes an approach to extend indenitely the duration of traction force measurements with cells cultured on soft polyacrylamide gels. Typical observation times in traction force measurements on similar substrates have been limited to 2448 h, but cell differentiation or responses to external stimuli often occur over much longer periods of several days or weeks. This study describes a method for covalently linking uorescent marker beads to a polyacrylamide matrix that renders the hydrogels useful for traction force measurements over several days. This approach was validated by comparing the contractility of C2C12 murine skeletal muscle cells prior to myotube formation, after one day in culture, with that of myotubes after 7 days in culture. Measured tractions increased concurrent with the differentiation of C2C12 cells to the contractile, myotube phenotype. Covalent bead linkage thus extends the useful period during which traction force data can be obtained with cells cultured on optically transparent polyacrylamide hydrogels with controlled elastic moduli. KeywordsTraction force microscopy, Polyacrylamide hydrogel, Cell contractility, Differentiation, Mechanosensing.

INTRODUCTION Microfabrication tools have been used extensively to investigate the impact of protein and small molecule concentration proles on cell behavior or cellular functions. In addition to soluble factors, adherent cells sense the chemical composition of the substrate via such cell surface receptors as integrins. These surface receptors also signal to regulate cellular functions such as adhesion, migration, or cell statefunctions that can be manipulated by controlling both substrate composition and ligand distribution. Despite substantial advances in the ability to control the identities, concentrations, and spatial distributions of specic, immobilized biochemical cues, until

Address correspondence to Deborah Leckband, Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801, USA. Electronic mail: leckband@illinois.edu

recently, this was always done with rigid substrates such as glass or polystyrene. These materials have elastic moduli on the order of 1100 GPa, while most tissues are much softer with elastic moduli on the order of 150 kPa.11,12 While in vitro experiments performed with cells cultured on glass or polystyrene have been informative models of in vivo cell behaviors, there is now extensive evidence that the substrate rigidity can dramatically alter cell functions. Since the rst demonstration that elastic substrata impact cell behavior, several publications documented the inuence of mechanical properties of cellular environments on cell functions.6,13,1820 Several researchers demonstrated that the use of appropriate substrate rigidity, in addition to chemical stimuli, enhances both the percentage of stem cells that differentiate to a particular lineage (e.g. mesenchymal stem cells,18 or neural stem cells19,20) and the cells ability to maintain that differentiated state.13 Engler et al.6 showed that culturing mesenchymal stem cells on collagen gels of different rigidity resulted in stem cells that express phenotypic and genotypic markers indicative of brain, muscle, or bone, depending on the substrate elastic modulus. Through integrin linkages to the matrix cells sense the substrate stiness and correspondingly modify cell properties.3 Some proteins in focal adhesions are tension sensors that probe the mechanical environment or sense the application of external force to trigger a cascade of reactions that reinforces the focal adhesion.24 Even without active external stimuli, cells generate contractile forces via the actomyosin cytoskeleton in order to grip the substrate. In addition to recruiting other proteins, focal adhesions activate signaling proteins such as small Rho GTPases that in turn modify contractile forces exerted on the substrate via the focal adhesions. Substrate deformations induced by these traction forces can be measured and translated into quantitative traction elds by a technique known as traction force microscopy.9 Traction force microscopy is based on the use of a deformable cell culture substrate, typically a hydrogel.
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The substrate is embedded with duciary markers, which are generally uorescent microbeads. Traction forces exerted by adherent cells generate small deformations in the gel that result in slight bead displacements. Using these displacements and knowledge of the elastic modulus and Poisson ratio of the hydrogel, one can calculate the traction forces exerted by the cells.22,23 Traction force measurements have typically been performed with cells in short-term culture (<48 h).1,9,14,17,22,23,26 However, there are cases in which longer term changes in cell behavior associated with, for example, differentiation, require observation over periods of several days. However, TFM is temporally limited because the entrained 0.2 lm uorescent marker beads diffuse out of the hydrogel matrix within a few days, thereby degrading the quality of traction force data over time. The use of larger beads would slow the diffusion, but would also diminish the spatial resolution of the traction force measurements. Micropilllars, which are also used for traction force measurements, do not suffer from this limitation.2,7 However, light scattering by pillar arrays interferes with imaging, and some argue that the spatial localization of adhesive proteins on the pillar tips does not represent ligand distributions in vivo. A solution to the temporal constraint of the TFM measurements, described in this work, is to covalently link the beads to the gel matrix. This prevents bead diffusion and retains traction force resolution in long-term studies. As a model system, we used the C2C12 murine skeletal muscle cell line, which is a widely used model for muscle cell dierentiation.10,15 These cells begin with a mononucleate, migratory, proliferative, myoblast phenotype, but are capable of differentiating into an elongated, multinucleate, myotube phenotype that is no longer migratory or proliferative, and that resembles the functional contractile unit of adult muscle.10,15 Differentiation requires myoblast fusion into multinucleate myotubes, a process triggered by serum starvation. Because this differentiation process requires several days in culture and relatively high cell densities, it is incompatible with current traction force measurement protocols. For successful traction force data collection, cells must be at a low density, but myoblast fusion requires cellcell contacts. To measure myotube contractile forces thus required a protocol that optimized these competing cell culture and TFM criteria. This report describes experimental approaches that enabled traction-force measurements of dierentiated myotubes after several days in culture. This requires the modication of both the substrate preparation and the C2C12 dierentiation protocols. The results, in which we demonstrate greater myotube contractility

relative to the initial myoblasts, provide experimental validation of these approaches.

RESULTS AND DISCUSSION Microbead Preparation Previously, traction force measurements were carried out within 2448 h of cell seeding on the elastic substrate. The entrapped beads are suciently stable in the gels to enable measurements over this time period. Figure 1 shows z-stack images of hydrogels in cell culture with either covalently linked or unlinked (entrapped) beads. Fluorescence intensity proles located below each image indicate uorescence intensity increases at bead locations, relative to the background. The TFM image analyses software locates bead positions based on the dierence in intensity relative to the background; therefore clear peaks in intensity are required for data analysis. However, even after just 1 day in culture, few beads remain on the top surface of the gel containing unlinked beads. Only by imaging deeper into the gel (20 lm below the surface) are distinct uorescent beads evident (arrows in Fig. 1). In contrast, gels with covalently bound beads had roughly the same number of beads (indicated by arrows) at each focal plane: namely, the top of the gel, 10 lm below the surface, and 20 lm below the surface. At day 6, the gels with linked beads retained a large number of beads at each focal plane, but there were few beads remaining in gels containing only physically entrapped beads. Additionally, the density of beads does not exhibit the same increase with depth as it did on day 1, indicating that beads also diffused out of the gel from greater depths. After 6 days the beads are fairly evenly, but sparsely distributed in the gel and oating above the gel in the culture medium. In contrast to the entrapped beads, covalently linked beads are retained in the gel, thereby ensuring sucient bead density for traction force resolution. This also decreases background uorescence from beads oating in the cell culture medium. Results in Fig. 1 demonstrate the success of the covalent bead-linking strategy. To achieve this, carboxy-terminated beads were activated to bind primary amines of acrylamide through 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)/N-hydroxy-succinimide ester (NHS) coupling chemistry.8 Initial tests showed that activated beads added directly to the gelation mixture interfered with the polymerization. This was avoided by rst activating beads with EDC/NHS. They were then suspended in acrylamide solution for 30 min (Fig. 2), and then rinsed thoroughly to remove unreacted reagents.

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FIGURE 1. Microbead loss from hydrogels after 6 days in culture medium. Phase contrast images show cells, in focus, on the gel surface (0 lm). Fluorescent images of the microbeads at this level (0 lm) as well as 10 and 20 lm below the gel surface show that in gels with linked beads, the bead density is similar at all focal planes of the gel at 1 or 6 days in culture. Fluorescent intensity proles (representing a line drawn diagonally from the top left to lower right corner of each image) indicate the presence of a bead by a sharp increase in intensity. The peaks which indicate beads that can be identied by the TFM programs are indicated by arrows. Gels with unlinked beads posses fewer beads (intensity peaks). Notes: (i) Image sections shown here are only fractions of actual traction force images. Space limitations prevent presentation of full size images at a resolution that allows identication of microbeads. The images shown here are only 1/25 of regular traction force images, this is why the cells are cut off in the phase contrast images. The image sections chosen and intensity proles are representative of the greater images. (ii) Filled circles indicate large intensity peaks made by clumps of microbeads, these are not analyzed by the TFM programs. (iii) Scale bar 30 lm.

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FIGURE 2. Reaction scheme for linking microbeads to the polyacrylamide gel matrix. Carboxy terminated beads are incubated in an EDC/NHS solution to allow the formation of the semi-stable amine reactive NHS ester. After rinsing, these beads are incubated in an acrylamide solution allowing the primary amine from the acrylamide to bind to the bead. The acrylamide-terminated beads co-polymerize with regular acrylamide monomers and bis-acrylamide monomers to form a polyacrylamide matrix containing covalently linked microbeads.

When the thus modied beads were added to the gelation mixture, polymerization proceeded as in the absence of the modied beads. Gel Characterization We determined the elastic moduli of polyacrylamide (5% w/v acrylamide) gels containing dierent amounts of bis-acrylamide cross-linker by analysis of the nanoindentation proles obtained with the atomic force microscope (AFM). The measured elastic moduli of 5% (w/v) acrylamide hydrogels ranged from 1.3 to 4.6 kPa, depending on the bis-acrylamide concentration (Fig. 3). To covalently link proteins to the hydrogel, polyacrylamide was copolymerized with acrylamidohexanoic acid NHS ester (N6), which was synthesized according to published procedures.16,21 The coupling chemistry is shown in Fig. 4, and experimental details are described in the Materials and Methods section. Comparing elastic moduli of the gels with and without the covalently coupled microbeads, showed

FIGURE 3. Elastic modulus dependence on bis-acrylamide concentration. Data are for 5 wt.% acrylamide gels with the indicated bis-acrylamide concentrations. Each point represents the average of 208 nano-indentation curves measured at 4 discrete locations on each of two hydrogels. Error bars represent one standard deviation from the mean.

that the bead linking did not alter the moduli of the gels. A gel with covalently bound microbeads had an elastic modulus of 2.8 0.5 kPa. The modulus of the gels prepared with the identical acrylamide/N6 linker/bis-acrylamide concentrations, but containing unbound beads, was 2.9 0.2 kPa. This indicates that covalent bead coupling had no statistically signicant

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impact on the mechanics (p = 0.32). Protein immobilization also did not affect the gel stiffness. For example, the elastic modulus of a 5% (w/v) acrylamide

gel with 0.1% (w/v) bis was 2.7 0.3 kPa, which is statistically the same as the 2.6 0.7 kPa measured after protein immobilization (p = 0.059). Traction Force Microscopy of Myoblasts and Dierentiated Myotubes C2C12 muscle cell contractility was measured both for myoblasts and for dierentiated myotubes in 6 day cultures. Figure 5 shows the difference between these two differentiation states. TFM analyses require low cell densities such that there is less than one cell per image frame. Otherwise, bead displacement elds associated with individual cells overlap, and this complicates analyses of the traction force maps generated by individual cells. This presents a particular challenge when using C2C12 cells because, in order for the fusion process to take placean essential step in myotube formationthe cells must be sufciently dense to encounter fusion partners. The protocol developed here optimized these two competing criteria (see Materials and Methods) and reliably produced myotubes of suitable length and sufciently low density for these analyses (Fig. 5b). A single, representative, data set is shown for each cell state: namely, myoblasts (Fig. 6) and myotubes (Fig. 7). These are typical of larger sample sizes. The myoblasts were imaged after 1 day in culture, whereas the myotubes were cultured for an additional 6 days prior to imaging (see Materials and Methods). The myotube (Fig. 7) has a projected area of 3700 lm2 compared to the myoblast area of 2200 lm2 (Fig. 6). This difference is characteristic of differentiated myotubes. As the gures show, the maximal displacement of the gel substrate beneath the myotube was about 1.2 lm, in contrast to only 0.7 lm for the myoblast. The average net contractile moment of myoblasts was 2.6 0.3 pJ (n = 3) while that of myotubes was 16.2 9 pJ (n = 3). These values are signicantly different (p = 0.11). Even the area-normalized contractile moments of 1.2 9 103 and 4.49 9 103pJ/lm2 for

FIGURE 4. N6 consists of an acrylamide functionality linked by a six-carbon chain to an NHS ester. It co-polymerizes with the polyacrylamide gel then amine couples proteins via the active ester.

FIGURE 5. Morphology of undifferentiated and differentiated C2C12 cells. (a) Undifferentiated C2C12 myoblasts. (b) A single, differentiated, C2C12 myotube at low culture density. Scale bars 10 lm.

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FIGURE 6. TFM images of a myoblast. (a) Phase contrast image of a single myoblast on a 2.6 kPa gel coated in laminin. (b) Displacement map indicating substrate deformation. (c) Unconstrained traction force map. (d) Constrained traction force map.

myoblasts and myotubes, respectively, are signicantly different. This transition to the myotube phenotype, which is similar to muscle formation in vivo, results in greater cell contractility as expected. It is important to emphasize that, while these results reveal intriguing dierences resulting from myotube formation, the objective of this study was to modify substrate preparation protocols to enable in-depth mechanistic studies of cellular changes during these long-term cultures. Importantly, the information in Figs. 6 and 7 could not have been obtained using standard gel preparation protocols for TFM substrata. Both the covalent linking of beads in the gels and optimizing cell culture and differentiation protocols enabled us to obtain these results.

CONCLUSIONS The results described demonstrate that previously described protocols for producing elastic substrates for

TFM measurements were insuciently robust for culture times greater than 48 h due to the loss of the marker particles from gels. The protocol described in this study for covalently linking the uorescent marker beads eectively stabilizes the bead/polyacrylamide hydrogel composite. This enabled the collection of quality traction force data for up to a week in culture on these transparent substrates, without altering the gel elastic modulus. TFM measurements of C2C12 cells cultured on laminin-coated polyacrylamide hydrogels indicate that myotube formation is accompanied by increased cell contractility, which is consistent with the physiological function of myotubes (muscle contraction) vs. myoblasts, which migrate to sites of muscle injury. The process of C2C12 cell dierentiation in vitro requires several days, but the stabilization of marker beads in gels was needed before such comparisons of myotube and myoblast contractility were possible. Although we validated the bead linking methodology with C2C12 cells, this modied TFM substrate protocol will be

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FIGURE 7. TFM images of a myotube. (a) Phase contrast image of a single myotube on a 2.6 kPa gel coated in laminin. (b) Displacement map indicating substrate deformation. (c) Unconstrained traction force map. (d) Constrained traction force map.

generally applicable to a wide range of investigations of the long-term evolution of cell contractility during, for example, differentiation or disease progression.

hydrochloride (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), aminopropyltriethoxysilane (APTES), and glutaraldehyde (grade I) were purchased from Sigma. Carboxy-terminated, rhodamine-labeled polystyrene beads were purchased from Invitrogen (formerly Molecular Probes). Cell Culture and Reagents C2C12 murine skeletal muscle cells were a gift from Prof. M. Boppart (University of Illlinois, Urbana). Cells were maintained in low-glucose DMEM supplemented with 10 vol.% fetal bovine serum (growth medium) or 2 vol.% horse serum (dierentiation medium), 1 vol.% penicillin/streptomycin and 1 vol.% L-glutamine (Hyclone). Cells were subcultured with the use of 19 trypsinEDTA (Hyclone). Cytosine b-D-arabinofuranoside hydrochloride (Ara-C) (Sigma) at a concentration of 10 lM in the differentiation medium was used to kill dividing cells.

MATERIALS AND METHODS Polyacrylamide Hydrogel Materials Acrylamide and cross-linker, NN-methylene-bisacrylamide, as well as the polymerization catalysts N,N,N,N-tetramethylene-diamine (TEMED) and ammonium persulfate were obtained from Bio-Rad. Carboxy terminated 0.2 lm rhodamine microbeads were obtained from Invitrogen. The N6 monomer was synthesized according to published procedures.16,21 Laminin was purchased from Invitrogen. Glass cover slips were from Corning. Tridecauoro-1-1-2-2-tetrahydrooctyl trichlorosilane (TTT) was purchased from Gelest. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide

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To generate low density myotubes for TFM studies, cell were initially plated at 1.5 9 104 cells per 6 cm Petri dish, left for 1 day in growth medium, and followed by 3 days in differentiation media and 2 days in kill medium (differentiation medium + Ara-C). Cells were then returned to the differentiation medium for 1 day before imaging. This complicated series of medium changes over 6 days optimized the number of short myotubes (23 nuclei) that were at sufciently low density for TFM measurements. C2C12 cells were plated in growth medium since plating in low serum differentiation medium causes mass necrosis. Three days in differentiation medium at low cell density allowed cells to form short myotubes (23 nuclei), and longer culture times resulted in longer myotubes. Two days in kill medium signicantly reduced the number of myoblasts remaining in the cultures. Myoblasts continued to apoptose during the nal day in differentiation medium.

Microbead Preparation Microbeads (2% solids, as purchased) were prepared in batches of 2001000 lL, as needed. Each aliquot was centrifuged to a pellet at 14000 rpm for 10 min in an Eppendorf Centrifuge 515C. Supernatant was removed, and beads were resuspended to original volume in EDC/Sulfo-NHS solution (3.8 and 7.6 mg/ mL, respectively) in ultrapure water. They were then incubated at room temperature for 30 min. Beads were rinsed once by spinning to a pellet, removing the supernatant, and then resuspending to the original volume in ultrapure water. Activated beads were then combined with an equal volume of acrylamide solution (10% w/v acrylamide in ultrapure water) and incubated at room temperature for 30 min. Finally, the acrylamide functionalized beads were rinsed three times in ultrapure water and resuspended to the original volume to remove remaining, unreacted reagents. Beads were stored in ultrapure water at 4 C and vortexed just prior to use in order to ensure a uniform suspension. Polyacrylamide Hydrogel Preparation Reaction solutions containing the desired amount of acrylamide and bis-acrylamide in ultrapure water were prepared. In this case 5% (w/v) acrylamide and 0.04 0.2% (w/v) bis-acrylamide were used. Microbeads were added at a concentration of 10 lL acrylamide modied beads (2% solids) per 1 mL gel solution. This fourcomponent solution was de-gassed under vacuum (~600 mm Hg) until bubbles failed to appear. Solutions of catalysts TEMED (10% w/v) and ammonium persulfate (10% w/v) were prepared in water. To initiate polymerization, 10 lL of each catalyst solution was added per 1 mL of gel solution. Finally, N6 linker was dissolved in cold ethanol at a concentration of 3 mg per 30 lL of ethanol for a 1 mL gel solution. This solution was triturated to disrupt N6 crystals, but they did not completely dissolve. The N6 concentration was optimized at 3 mg/mL gel solution (data not shown), and this concentration was used in all gels described in this study. TEMED, ammonium persulfate and N6 were added to the degassed gel solution immediately before plating the reaction mixture onto the glutaraldehyde activated cover slips. The nal gel solution contained 1 mL of the acrylamide/bisacrylamide/beads/water mixture plus 10 lL TEMED solution, 10 lL ammonium persulfate solution and 30 lL N6 solution. After thorough mixing, 50 lL of the nal gel solution was added to each glutaraldehyde activated glass cover slip and then covered with a silanized #1 22 9 22 mm cover slip to spread the gel. With this volume of solution, the set gels are ~100 lm thick. After allowing the gels to polymerize at room

Covalent Incorporation of Fluorescent Microbeads in Polyacrylamide Hydrogels Polyacrylamide hydrogels were fabricated using a modication of published protocols.25 These modications, described below, covalently linked microbeads in the gels. Glass Preparation Glass cover slips used to atten the gels and protect them from air during polymerization were prepared as follows. Briey, #1 22 9 22 mm cover slips were coated with TTT by vapor deposition. The slides were placed in a vacuum chamber together with a container with a few drops of TTT, and the pressure was reduced by ~600 mm Hg and left overnight at room temperature. The uorinated silane (TTT) forms a self-assembled monolayer on the glass, and thus produces a nonstick surface coating. The glass supports for the hydrogels were #1.5 22 9 40 mm cover slips that were cleaned in piranha solution (3:1 (v/v) sulfuric acid:hydrogen peroxide) for 45 min at room temperature, then rinsed thoroughly with ultrapure water (18 MX resistance). Clean cover slips were covalently modied with amines, to enhance polyacrylamide adhesion to the glass slides, by coating them with pure APTES for 30 min at room temperature. After the reaction, the slides were rinsed with ultrapure water, and left on the bench to air dry. They were then baked 30 min at 110 C to drive o any remaining water. Slides were then coated with 0.5% (v/v) glutaraldehyde in water for 30 min, rinsed with ultrapure water, and allowed to air dry before use. The glutaraldehyde enables covalent bonding between the acrylamide gels and glass slides.

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temperature for 45 min, the top, silanized, cover slips were removed. Laminin is the ligand for the a7b1 integrin expressed by muscle cells, and is the matrix protein on substrates used in these experiments. Each gel was therefore covered in 200 lL of laminin at 0.1 mg/mL in Dulbeccos Phosphate Buffered Saline (DPBS), at pH 7.2. After allowing the protein to react with the NHS groups of N6 in the gel for 1 h at room temperature, the gels were stored at 4 C overnight. Cells were plated on the gels on the day following the gel preparation. Excess protein solution was allowed to drain from the gels before use. To seed the cells onto the gels, the #1.5 cover slips supporting the gels were placed in individual 6 cm diameter Petri dishes. The cover slips with the covalently bound gels were axed to the Petri dish with 100% silicone sealant (GE). The gels were then equilibrated with cell culture medium containing 1% penicillin/streptomycin at 37 C for 30 min prior to seeding cells. Hydrogel Stiness Characterization Hydrogel elastic moduli (Youngs moduli) were determined by nanoindentation using an atomic force microscope (Asylum). The force-distance curves were analyzed using the following model, as described previously.4,5 s kd d0 z z 0 d d0 2=pE=1 m2 tan a where z = the cantilever height, d = the tip deection, k = the spring constant of the tip, E = the elastic modulus of the sample (to be calculated), a = the tip half opening angle, m = the Poisson ratio of the sample. AFM tips (silicon nitride, MLCT) were obtained from Veeco Probes with a half opening angle of 17, and the gels were assumed to have a Poisson ratio of 0.5.4 Traction Force Measurements Cells were imaged for traction force measurements on a Zeiss Axiovert 100 M using Axiovision 4.6 software. Single cells (with no near neighbors) were located on the hydrogel surface and imaged in both phase contrast (to see the cell outline) and uorescence (to obtain the rhodamine microbead positions). To obtain the traction-free bead image, cells were released from the gels at the end of the TFM studies by adding 1 mL of 1% (w/v) SDS (Fisher) to the culture (5 mL culture medium). Cells completely dissolved within a few minutes, and the phase and uorescence imaging was

repeated. The before and after bead images were analyzed by Fourier Transform Traction Cytometry (FTTC) programs provided by Prof. N. Wang (Mechanical Engineering, University of Illinois, Urbana).

ACKNOWLEDGMENTS This work was supported by the Reid T. Milner Professorship to DEL and CM was supported by 5 P01 HL060678. The authors have no nancial interests in this work.

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