Cell motility and local viscoelasticity of fibroblasts

Viscoelastic changes of the lamellipodial actin cytoskeleton are a fundamental element of cell motility. Thus, the correlation between the local viscoelastic properties of the lamellipodium (including the transitional region to the cell body) and the speed of lamellipodial extension is studied for normal and malignantly transformed fibroblasts. Using our atomic force microscopy-based microrheology technique, we found different mechanical properties between the lamellipodia of malignantly transformed fibroblasts (H-ras transformed and SV-T2 fibroblasts) and normal fibroblasts (BALB 3T3 fibroblasts). The average elastic constants, K, in the leading edge of SV-T2 fibroblasts (0.48 +/- 0.51 kPa) and of H-ras transformed fibroblasts (0.42 +/- 0.35 kPa) are significantly lower than that of BALB 3T3 fibroblasts (1.01 +/- 0.40 kPa). The analysis of time-lapse phase contrast images shows that the decrease in the elastic constant, K, for malignantly transformed fibroblasts is correlated with the enhanced motility of the lamellipodium. The measured mean speeds are 6.1 +/- 4.5 microm/h for BALB 3T3 fibroblasts, 13.1 +/- 5.2 microm/h for SV-T2 fibroblasts, and 26.2 +/- 11.5 microm/h for H-ras fibroblasts. Furthermore, the elastic constant, K, increases toward the cell body in many instances which coincide with an increase in actin filament density toward the cell body. The correlation between the enhanced motility and the decrease in viscoelastic moduli supports the Elastic Brownian Ratchet model for driving lamellipodia extension.     

Local rheology of human neutrophils investigated using atomic force microscopy

During the immune response, neutrophils display localized mechanical events by interacting with their environment through the micro-vascular transit, trans-endothelial, and trans-epithelial migration. Nano-mechanical studies of human neutrophils on localized nano-domains could provide the essential information for understanding their immune responsive functions. Using the Atomic Force Microscopy (AFM)-based micro-rheology, we have investigated rheological properties of the adherent human neutrophils on local nano-domains. We have applied the modified Hertz model to obtain the viscoelastic moduli from the relatively thick body regions of the neutrophils. In addition, by using more advanced models to account for the substrate effects, we have successfully characterized the rheological properties of the thin leading and tail regions as well. We found a regional difference in the mechanical compliances of the adherent neutrophils. The central regions of neutrophils were significantly stiffer (1,548 ± 871 Pa) than the regions closer to the leading edge (686 ± 801 Pa), while the leading edge and the tail (494 ± 537 Pa) regions were mechanically indistinguishable. The frequency-dependent elastic and viscous moduli also display a similar regional difference. Over the studied frequency range (100 to 300 Hz), the complex viscoelastic moduli display the partial rubber plateau behavior where the elastic moduli are greater than the viscous moduli for a given frequency. The non-disparaging viscous modulus indicates that the neutrophils display a viscoelastic dynamic behavior rather than a perfect elastic behavior like polymer gels. In addition, we found no regional difference in the structural damping coefficient between the leading edge and the cell body. Thus, we conclude that despite the lower loss and storage moduli, the leading edges of the human neutrophils display partially elastic properties similar to the cell body. These results suggest that the lower elastic moduli in the leading edges are more favorable for the elastic fluctuation of actin filaments, which supports the polymerization of the actin filaments leading to the active protrusion during the immune response.     

Unconfined creep compression of chondrocytes

The study of single cell mechanics offers a valuable tool for understanding cellular milieus. Specific knowledge of chondrocyte biomechanics could lead to elucidation of disease etiologies and the biomechanical factors most critical to stimulating regenerative processes in articular cartilage. Recent studies in our laboratory have suggested that it may be acceptable to approximate the shape of a single chondrocyte as a disc. This geometry is easily utilized for generating models of unconfined compression. In this study, three continuum mechanics models of increasing complexity were formulated and used to fit unconfined compression creep data. Creep curves were obtained from middle/deep zone chondrocytes (n = 15) and separately fit using the three continuum models. The linear elastic solid model yielded a Young's modulus of 2.55+/-0.85 kPa. The viscoelastic model (adapted from the Kelvin model) generated an instantaneous modulus of 2.47+/-0.85 kPa, a relaxed modulus of 1.48+/-0.35 kPa, and an apparent viscosity of 1.92+/-1.80 kPa-s. Finally, a linear biphasic model produced an aggregate modulus of 2.58+/-0.87 kPa, a permeability of 2.57 x 10(-12)+/-3.09 m(4)/N-s, and a Poisson's ratio of 0.069+/-0.021. The results of this study demonstrate that similar values for the cell modulus can be obtained from three models of increasing complexity. The elastic model provides an easy method for determining the cell modulus, however, the viscoelastic and biphasic models generate additional material properties that are important for characterizing the transient response of compressed chondrocytes.     

Viscoelastic properties of single attached cells under compression

The viscoelastic properties of single, attached C2C12 myoblasts were measured using a recently developed cell loading device. The device allows global compression of an attached cell, while simultaneously measuring the associated forces. The viscoelastic properties were examined by performing a series of dynamic experiments over two frequency decades (0.1-10 Hz) and at a range of axial strains (approximately 10-40%). Confocal laser scanning microscopy was used to visualize the cell during these experiments. To analyze the experimentally obtained force-deformation curves, a nonlinear viscoelastic model was developed. The nonlinear viscoelastic model was able to describe the complete series of dynamic experiments using only a single set of parameters, yielding an elastic modulus of 2120 +/- 900 Pa for the elastic spring, an elastic modulus of 1960 +/- 1350 for the nonlinear spring, and a relaxation time constant of 0.3 +/- 0.12 s. To our knowledge, it is the first time that the global viscoelastic properties of attached cells have been quantified over such a wide range of strains. Furthermore, the experiments were performed under optimal environmental conditions and the results are, therefore, believed to reflect the viscoelastic mechanical behavior of cells, such as would be present in vivo.     

Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry.

A magnetic bead microrheometer has been designed which allows the generation of forces up to 10(4) pN on 4.5 micron paramagnetic beads. It is applied to measure local viscoelastic properties of the surface of adhering fibroblasts. Creep response and relaxation curves evoked by tangential force pulses of 500-2500 pN (and approximately 1 s duration) on the magnetic beads fixed to the integrin receptors of the cell membrane are recorded by particle tracking. Linear three-phasic creep responses consisting of an elastic deflection, a stress relaxation, and a viscous flow are established. The viscoelastic response curves are analyzed in terms of a series arrangement of a dashpot and a Voigt body, which allows characterization of the viscoelastic behavior of the adhering cell surface in terms of three parameters: an effective elastic constant, a viscosity, and a relaxation time. The displacement field generated by the local tangential forces on the cell surface is visualized by observing the induced motion of assemblies of nonmagnetic colloidal probes fixed to the membrane. It is found that the displacement field decays rapidly with the distance from the magnetic bead. A cutoff radius of Rc approximately 7 micron of the screened elastic field is established. Partial penetration of the shear field into the cytoplasm is established by observing the induced deflection of intracellular compartments. The cell membrane was modeled as a thin elastic plate of shear modulus mu * coupled to a viscoelastic layer, which is fixed to a solid support on the opposite side; the former accounts for the membrane/actin cortex, and the latter for the contribution of the cytoskeleton to the deformation of the cell envelope. It is characterized by the coupling constant chi characterizing the elasticity of the cytoskeleton. The coupling constant chi and the surface shear modulus mu * are obtained from the measured displacements of the magnetic and nonmagnetic beads. By analyzing the experimental data in terms of this model a surface shear modulus of mu * approximately 2 . 10(-3) Pa m to 4 . 10(-3) Pa m is found. By assuming an approximate plate thickness of 0.1 micron one estimates an average bulk shear modulus of mu approximately (2 / 4) . 10(-4) Pa, which is in reasonable agreement with data obtained by atomic force microscopy. The viscosity of the dashpot is related to the apparent viscosity of the cytoplasm, which is obtained by assuming that the top membrane is coupled to the bottom (fixed) membrane by a viscous medium. By application of the theory of diffusion of membrane proteins in supported membranes we find a coefficient of friction of bc approximately 2 . 10(9) Pa s/m corresponding to a cytoplasmic viscosity of 2 . 10(3) Pa s.     

Viscoelastic properties of f-actin, microtubules, f-actin/alpha-actinin, and f-actin/hexokinase determined in microliter volumes with a novel nondestructive method

A nondestructive method to determine viscoelastic properties of gels and fluids involves an oscillating glass fiber serving as a sensor for the viscosity of the surrounding fluid. Extremely small displacements (typically 1-100 nm) are caused by the glass rod oscillating at its resonance frequency. These displacements are analyzed using a phase-sensitive acoustic microscope. Alterations of the elastic modulus of a fluid or gel change the propagation speed of a longitudinal acoustic wave. The system allows to study quantities as small as 10 microliters with temporal resolution >1 Hz. For 2-100 microM f-actin gels a final viscosity of 1.3-9.4 mPa s and a final elastic modulus of 2.229-2.254 GPa (corresponding to 1493-1501 m/s sound velocity) have been determined. For 10- to 100-microM microtubule gels (native, without stabilization by taxol), a final viscosity of 1.5-124 mPa s and a final elastic modulus of 2.288-2. 547 GPa (approximately 1513-1596 m/s) have been determined. During polymerization the sound velocity in low-concentration actin solutions increased up to +1.3 m/s (approximately 1.69 kPa) and decreased up to -7 m/s (approximately 49 kPa) at high actin concentrations. On polymerization of tubulin a concentration-dependent decrease of sound velocity was observed, too (+48 to -12 m/s approximately 2.3-0.1 MPa, for 10- to 100-microM tubulin). This decrease was interpreted by a nematic phase transition of the actin filaments and microtubules with increasing concentration. 2 mM ATP (when compared to 0.2 mM ATP) increased polymerization rate, final viscosity and elastic modulus of f-actin (17 microM). The actin-binding glycolytic enzyme hexokinase also accelerated the polymerization rate and final viscosity but elastic modulus (2.26 GPa) was less than for f-actin polymerized in presence of 0.2 mM ATP (2.28 GPa).     

Elastic wrinkling of keratocyte lamellipodia driven by myosin-induced contractile stress

During actin-based cell migration, the actin cytoskeleton in the lamellipodium both generates and responds to force, which has functional consequences for the ability of the cell to extend protrusions. However, the material properties of the lamellipodial actin network and its response to stress on the timescale of motility are incompletely understood. Here, we describe a dynamic wrinkling phenotype in the lamellipodium of fish keratocytes, in which the actin sheet buckles upward away from the ventral membrane of the cell, forming a periodic pattern of wrinkles perpendicular to the cell’s leading edge. Cells maintain an approximately constant wrinkle wavelength over time despite new wrinkle formation and the lateral movement of wrinkles in the cell frame of reference, suggesting that cells have a preferred or characteristic wrinkle wavelength. Generation of wrinkles is dependent upon myosin contractility, and their wavelength scales directly with the density of the actin network and inversely with cell adhesion. These results are consistent with a simple physical model for wrinkling in an elastic sheet under compression and suggest that the lamellipodial cytoskeleton behaves as an elastic material on the timescale of cell migration despite rapid actin turnover.     

Creep indentation of single cells

An apparatus for creep indentation of individual adherent cells was designed, developed, and experimentally validated. The creep cytoindentation apparatus (CCA) can perform stress-controlled experiments and measure the corresponding deformation of single anchorage-dependent cells. The apparatus can resolve forces on the order of 1 nN and cellular deformations on the order of 0.1 micron. Experiments were conducted on bovine articular chondrocytes using loads on the order of 10 nN. The experimentally observed viscoelastic behavior of these cells was modeled using the punch problem and standard linear solid. The punch problem yielded a Young's modulus of 1.11 +/- 0.48 kPa. The standard linear solid model yielded an instantaneous elastic modulus of 8.00 +/- 4.41 kPa, a relaxed modulus of 1.09 +/- 0.54 kPa, an apparent viscosity of 1.50 +/- 0.92 kPa-s, and a time constant of 1.32 +/- 0.65 s. To our knowledge, this is the first time that stress-controlled indentation testing has been applied at the single cell level. This methodology represents a new tool in understanding the mechanical nature of anchorage-dependent cells and mechanotransductional pathways.     

Viscoelastic characterization of the porcine temporomandibular joint disc under unconfined compression

Pathophysiology of the temporomandibular joint (TMJ) disc is central to many orofacial disorders; however, mechanical characterization of this tissue is incomplete. In this study, we identified surface-regional mechanical variations in the porcine TMJ disc under unconfined compression. The intermediate zone, posterior, anterior, lateral, and medial regions of eight TMJ discs were sectioned into inferior and superior surface samples. Surface-regional sections were then subjected to incremental stress relaxation tests. Single strain step (SSS) and final deformation (FD) viscoelastic models were fit to experimental data. Both models represented the experimental data with a high degree of accuracy (R(2)=0.93). The instantaneous modulus and relaxation modulus for the TMJ disc sections were approximately 500 kPa and 80 kPa, respectively; the coefficient of viscosity was approximately 3.5 MPa-s. Strain dependent material properties were observed across the disc's surface-regions. Regional variations in stiffness were observed in both models. The relaxation modulus was largest in the inferior-medial parts of the disc. The instantaneous modulus was largest in the posterior and anterior regions of the disc. Surface-to-surface variations were observed in the relaxation modulus for only the FD model; the inferior surface was found to be more resistant to compression than the superior surface. The results of this study imply the stiffness of the TMJ disc may change as strain is applied. Furthermore, the lateral region exhibited a lower viscosity and stiffness compared to other disc regions. Both findings may have important implications on the TMJ disc's role in jaw motion and function.     

Determination of mechanical properties of soft tissue scaffolds by atomic force microscopy nanoindentation

While the determination of mechanical properties of a hard scaffold is relatively straightforward, the mechanical testing of a soft tissue scaffold poses significant challenges due in part to its fragility. Here, we report a new approach for characterizing the stiffness and elastic modulus of a soft scaffold through atomic force microscopy (AFM) nanoindentation. Using collagen-chitosan hydrogel scaffolds as model soft tissue scaffolds, we demonstrated the feasibility of using AFM nanoindentation to determine a force curve of a soft tissue scaffold. A mathematical model was developed to ascertain the stiffness and elastic modulus of a scaffold from its force curve obtained under different conditions. The elastic modulus of a collagen-chitosan (80%/20%, v/v) scaffold is found to be 3.69 kPa. The scaffold becomes stiffer if it contains more chitosan. The elastic modulus of a scaffold composed of 70% collagen and 30% chitosan is about 11.6 kPa. Furthermore, the stiffness of the scaffold is found to be altered significantly by extracellular matrix deposited from cells that are grown inside the scaffold. The elastic modulus of collagen-chitosan scaffolds increased from 10.5 kPa on day 3 to 63.4 kPa on day 10 when human foreskin fibroblast cells grew inside the scaffolds. Data acquired from these measurements will offer new insights into understanding cell fate regulation induced by physiochemical cues of tissue scaffolds.     

Combining AFM and Acoustic Probes to Reveal Changes in the Elastic Stiffness Tensor of Living Cells

Knowledge of how the elastic stiffness of a cell affects its communication with its environment is of fundamental importance for the understanding of tissue integrity in health and disease. For stiffness measurements, it has been customary to quote a single parameter quantity, e.g., Young’s modulus, rather than the minimum of two terms of the stiffness tensor required by elasticity theory. In this study, we use two independent methods (acoustic microscopy and atomic force microscopy nanoindentation) to characterize the elastic properties of a cell and thus determine two independent elastic constants. This allows us to explore in detail how the mechanical properties of cells change in response to signaling pathways that are known to regulate the cell’s cytoskeleton. In particular, we demonstrate that altering the tensioning of actin filaments in NIH3T3 cells has a strong influence on the cell's shear modulus but leaves its bulk modulus unchanged. In contrast, altering the polymerization state of actin filaments influences bulk and shear modulus in a similar manner. In addition, we can use the data to directly determine the Poisson ratio of a cell and show that in all cases studied, it is less than, but very close to, 0.5 in value.     

Combining AFM and Acoustic Probes to Reveal Changes in the Elastic Stiffness Tensor of Living Cells

Knowledge of how the elastic stiffness of a cell affects its communication with its environment is of fundamental importance for the understanding of tissue integrity in health and disease. For stiffness measurements, it has been customary to quote a single parameter quantity, e.g., Young’s modulus, rather than the minimum of two terms of the stiffness tensor required by elasticity theory. In this study, we use two independent methods (acoustic microscopy and atomic force microscopy nanoindentation) to characterize the elastic properties of a cell and thus determine two independent elastic constants. This allows us to explore in detail how the mechanical properties of cells change in response to signaling pathways that are known to regulate the cell’s cytoskeleton. In particular, we demonstrate that altering the tensioning of actin filaments in NIH3T3 cells has a strong influence on the cell''s shear modulus but leaves its bulk modulus unchanged. In contrast, altering the polymerization state of actin filaments influences bulk and shear modulus in a similar manner. In addition, we can use the data to directly determine the Poisson ratio of a cell and show that in all cases studied, it is less than, but very close to, 0.5 in value.     

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) approximately 2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-microm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 microm but increased significantly between 10 and 15 microm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.     

Mechanics of living cells measured by laser tracking microrheology

To establish laser-tracking microrheology (LTM) as a new technique for quantifying cytoskeletal mechanics, we measure viscoelastic moduli with wide bandwidth (5 decades) within living cells. With the first subcellular measurements of viscoelastic phase angles, LTM provides estimates of solid versus liquid behavior at different frequencies. In LTM, the viscoelastic shear moduli are inferred from the Brownian motion of particles embedded in the cytoskeletal network. Custom laser optoelectronics provide sub-nanometer and near-microsecond resolution of particle trajectories. The kidney epithelial cell line, COS7, has numerous spherical lipid-storage granules that are ideal probes for noninvasive LTM. Although most granules are percolating through perinuclear spaces, a subset of perinuclear granules is embedded in dense viscoelastic cytoplasm. Over all time scales embedded particles exhibit subdiffusive behavior and are not merely tethered by molecular motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2)) are more rigid than viscoelastic perinuclear regions (330 +/- 250 dyne/cm(2), p < 0.0001), but spectra converge at high frequencies. Although the actin-disrupting agent, latrunculin A, softens and liquefies lamellae, physiological levels of F-actin, alone (11 +/- 1.2 dyne/cm(2)) are approximately 70-fold softer than lamellae. Therefore, F-actin is necessary for lamellae mechanics, but not sufficient. Furthermore, in time-lapse of apparently quiescent cells, individual lamellar granules can show approximately 4-fold changes in moduli that last >10 s. Over a broad range of frequencies (0.1-30, 000 rad/s), LTM provides a unique ability to noninvasively quantify dynamic, local changes in cell viscoelasticity.     

Stresses at the cell-to-substrate interface during locomotion of fibroblasts

Recent technological improvements in the elastic substrate method make it possible to produce spatially resolved measurements of the tractions exerted by single motile cells. In this study we have applied these developments to produce maps of the tractions exerted by 3T3 fibroblasts during steady locomotion. The resulting images have a spatial resolution of approximately 5 micrometers and a maximum intensity of approximately 10(2) kdyn/cm2 (10(4) pN/micrometers2). We find that the propulsive thrust for fibroblast locomotion, approximately 0.2 dyn, is imparted to the substratum within 15 micrometers of the leading edge. These observations demonstrate that the lamellipodium of the fibroblast is able to generate intense traction stress. The cell body and posterior seem to be mechanically passive structures pulled forward entirely by this action.     

Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.     

Deformation properties of articular chondrocytes: a critique of three separate techniques

This paper presents a series of techniques, which examine the deformation characteristics of bovine articular chondrocytes. The direct contact approach employs well established methodology, involving AFM and micropipette aspiration, to yield structural properties of local regions of isolated chondrocytes. The former technique yields a non-linear response with increased structural stiffness in a central location on a projected image of the chondrocyte. A simple viscoelastic model can be used with data from the micropipette aspiration technique to yield a mean value of Young's modulus, which is similar to that recently reported (Jones et al., 1999). An indirect approach is also described, involving the response of chondrocytes seeded within compressed agarose constructs. For 1% agarose constructs, the resulting cell strain, yields a gross cell modulus of 2.7 kPa. The study highlights the difficulties in establishing unique mechanical parameters, which reflect the deformation behaviour of articular chondrocytes.     

Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells

This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force-indentation curves to an elastic model yields an elastic modulus of 7.22 +/- 0. 46 kPa over the nucleus, 2.97 +/- 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 +/- 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p < 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after force application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface.     

Development of Polydimethylsiloxane Substrates with Tunable Elastic Modulus to Study Cell Mechanobiology in Muscle and Nerve

Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS) blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line) and C2C12 (muscle cell line) were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.