A mechanics theory for the exploration of a high-throughput, sterile 3D in vitro traumatic brain injury model.

Brain injuries resulting from mechanical trauma represent an ongoing global public health issue. Several in vitro and in vivo models for traumatic brain injury (TBI) continue to be developed for delineating the various complex pathophysiological processes involved in its onset and progression. Developing an in vitro TBI model that is based on cortical spheroids is especially of great interest currently because they can replicate key aspects of in vivo brain tissue, including its electrophysiology, physicochemical microenvironment, and extracellular matrix composition. Being able to mechanically deform the spheroids are a key requirement in any effective in vitro TBI model. The spheroids' shape and size, however, make mechanically loading them, especially in a high-throughput, sterile, and reproducible manner, quite challenging. To address this challenge, we present an idea for a spheroid-based, in vitro TBI model in which the spheroids are mechanically loaded by being spun by a centrifuge. (An experimental demonstration of this new idea will be published shortly elsewhere.) An issue that can limit its utility and scope is that imaging techniques used in 2D and 3D in vitro TBI models cannot be readily applied in it to determine spheroid strains. In order to address this issue, we developed a continuum mechanics-based theory to estimate the spheroids' strains when they are being spun at a constant angular velocity. The mechanics theory, while applicable here to a special case of the centrifuge-based TBI model, is also of general value since it can help with the further exploration and development of TBI models.

Cortical spheroids show strain-dependent cell viability loss and neurite disruption following sustained compression injury.

Sustained compressive injury (SCI) in the brain is observed in numerous injury and pathological scenarios, including tumors, ischemic stroke, and traumatic brain injury-related tissue swelling. Sustained compressive injury is characterized by tissue loading over time, and currently, there are few in vitro models suitable to study neural cell responses to strain-dependent sustained compressive injury. Here, we present an in vitro model of sustained compressive neural injury via centrifugation. Spheroids were made from neonatal rat cortical cells seeded at 4000 cells/spheroid and cultured for 14 days in vitro. A subset of spheroids was centrifuged at 104, 209, 313 or 419 rads/s for 2 minutes. Modeling the physical deformation of the spheroids via finite element analyses, we found that spheroids centrifuged at the aforementioned angular velocities experienced pressures of 10, 38, 84 and 149 kPa, respectively, and compressive (resp. tensile) strains of 10% (5%), 18% (9%), 27% (14%) and 35% (18%), respectively. Quantification of LIVE-DEAD assay and Hoechst 33342 nuclear staining showed that centrifuged spheroids subjected to pressures above 10 kPa exhibited significantly higher DNA damage than control spheroids at 2, 8, and 24 hours post-injury. Immunohistochemistry of ß3-tubulin networks at 2, 8, and 24 hours post-centrifugation injury showed increasing degradation of microtubules over time with increasing strain. Our findings show that cellular injuries occur as a result of specific levels and timings of sustained tissue strains. This experimental SCI model provides a high throughput in vitro platform to examine cellular injury, to gain insights into brain injury that could be targeted with therapeutic strategies.

Topography, cell response, and nerve regeneration.

In the body, cells encounter a complex milieu of signals, including topographical cues, in the form of the physical features of their surrounding environment. Imposed topography can affect cells on surfaces by promoting adhesion, spreading, alignment, morphological changes, and changes in gene expression. Neural response to topography is complex, and it depends on the dimensions and shapes of physical features. Looking toward repair of nerve injuries, strategies are being explored to engineer guidance conduits with precise surface topographies. How neurons and other cell types sense and interpret topography remains to be fully elucidated. Studies reviewed here include those of topography on cellular organization and function as well as potential cellular mechanisms of response.

Cortical spheroids display oscillatory network dynamics.

Three-dimensional brain cultures can facilitate the study of central nervous system function and disease, and one of the most important components that they present is neuronal activity on a network level. Here we demonstrate network activity in rodent cortical spheroids while maintaining the networks intact in their 3D state. Networks developed by nine days in culture and became more complex over time. To measure network activity, we imaged neurons in rat and mouse spheroids labelled with a calcium indicator dye, and in mouse spheroids expressing GCaMP. Network activity was evident when we electrically stimulated spheroids, was abolished with glutamatergic blockade, and was altered by GABAergic blockade or partial glutamatergic blockade. We quantified correlations and distances between somas with micron-scale spatial resolution. Spheroids seeded at as few as 4000 cells gave rise to emergent network events, including oscillations. These results are the first demonstration that self-assembled rat and mouse spheroids exhibit network activity consistent with in vivo network events. These results open the door to experiments on neuronal networks that require fewer animals and enable high throughput experiments on network-perturbing alterations in neurons and glia.

Human neurons from Christianson syndrome iPSCs reveal mutation-specific responses to rescue strategies.

Christianson syndrome (CS), an X-linked neurological disorder characterized by postnatal attenuation of brain growth (postnatal microcephaly), is caused by mutations in SLC9A6, the gene encoding endosomal Na+/H+ exchanger 6 (NHE6). To hasten treatment development, we established induced pluripotent stem cell (iPSC) lines from patients with CS representing a mutational spectrum, as well as biologically related and isogenic control lines. We demonstrated that pathogenic mutations lead to loss of protein function by a variety of mechanisms: The majority of mutations caused loss of mRNA due to nonsense-mediated mRNA decay; however, a recurrent, missense mutation (the G383D mutation) had both loss-of-function and dominant-negative activities. Regardless of mutation, all patient-derived neurons demonstrated reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Phenotype rescue strategies showed mutation-specific responses: A gene transfer strategy was effective in nonsense mutations, but not in the G383D mutation, wherein residual protein appeared to interfere with rescue. In contrast, application of exogenous trophic factors (BDNF or IGF-1) rescued arborization phenotypes across all mutations. These results may guide treatment development in CS, including gene therapy strategies wherein our data suggest that response to treatment may be dictated by the class of mutation.

The importance of defining 'data' in data management policies: Commentary on: "Issues in data management".

What comprises 'data' varies from one institution to another based on the information which is deemed important by individual institutions. To effectively and efficiently produce, collect, and retain data, an organization develops specific defining characteristics of data to meet its informational needs. Procedures to maintain and retain knowledge among laboratory members and principal investigators will allow for improved efficiency of data collection. Optimization of communication, maintenance of inventories, record keeping, and updating relevant training programs are all critical to supporting the quality and integrity of a particular organization's data. Concurrent revisions to such procedures will ensure that the definition of data as well as the means by which it is collected and maintained remain appropriate to the needs of the individual organization.

Neurite outgrowth at the interface of 2D and 3D growth environments.

Growing neurons navigate complex environments, but in vitro systems for studying neuronal growth typically limit the cues to flat surfaces or a single type of cue, thereby limiting the resulting growth. Here we examined the growth of neurons presented with two-dimensional (2D) substrate-bound cues when these cues were presented in conjunction with a more complex three-dimensional (3D) architecture. Dorsal root ganglia (DRG) explants were cultured at the interface between a collagen I matrix and a glass coverslip. Laminin (LN) or chondroitin sulfate proteoglycans (CSPG) were uniformly coated on the surface of the glass coverslip or patterned in 50 microm tracks by microcontact printing. Quantitative analysis of neurite outgrowth with a novel grid system at multiple depths in the gel revealed several interesting trends. Most of the neurites extended at the surface of the gel when LN was presented whereas more neurites extended into the gel when CSPG was presented. Patterning of cues did not affect neurite density or depth of growth. However, neurite outgrowth near the surface of the gel aligned with LN patterns, and these extensions were significantly longer than neurites extended in other cultures. In interface cultures, DRG growth patterns varied with the type of cue where neurite density was higher in cultures presenting LN than in cultures presenting CSPG. These results represent an important step toward understanding how neurons integrate local structural and chemical cues to make net growth decisions.

Dynactin is essential for growth cone advance.

Dynactin is a multi-subunit complex that serves as a critical cofactor of the microtubule motor cytoplasmic dynein. We previously identified dynactin in the nerve growth cone. However, the function of dynactin in the growth cone is still unclear. Here we show that dynactin in the growth cone is required for constant forward movement of the growth cone. Chromophore-assisted laser inactivation (CALI) of dynamitin, a dynactin subunit, within the growth cone markedly decreases the rate of growth cone advance. CALI of dynamitin in vitro dissociates another dynactin subunit, p150(Glued), from dynamitin. These results indicate that dynactin, especially the interaction between dynamitin and p150(Glued), plays an essential role in growth cone advance.

Evaluation of neurite outgrowth anisotropy using a novel application of circular analysis.

Precise axon growth is required for making proper connections in development and after injury. One method of studying axon guidance and growth is through in vitro outgrowth assays that present controlled microenvironments. In this study, we applied circular statistical methods to evaluate directional neurite response. Visualization of data on a circular scale allows more accurate representation of the data, as neurite angles are inherently expressed on a circle. Here, the direction of neurite outgrowth from dorsal root ganglia derived neurons on different substrate types was quantitatively measured. Further, simulations of datasets with known circular parameters reflecting expected neurite angle distributions from different substrate types were also generated. Circular statistical methods were utilized and compared to linear statistical models widely used in the neuroscience literature. For small samples, Rao's spacing test showed the smallest occurrence of Type I errors (false positives) when tested against simulated uniform distributions. V-test and Rayleigh's test showed highest statistical power when tested against a unimodal distribution with known and unknown mean direction, respectively. For bimodal samples, Watson's U(2)-test showed the highest statistical power. Overall, circular statistical uniformity tests showed higher statistical power than linear non-parametric tests, particularly for small samples (n=5). Circular analysis methods represent a useful tool for evaluation of directionality of neurite outgrowth with applications including: (1) assessment of neurite outgrowth potential; (2) determination of isotropy of cellular responses to single and multiple cues and (3) determination of the relative strengths of cues present in a complex environment.

Schwann cell durotaxis can be guided by physiologically relevant stiffness gradients.

BACKGROUND: Successful nerve regeneration depends upon directed migration of morphologically specialized repair state Schwann cells across a nerve defect. Although several groups have studied directed migration of Schwann cells in response to chemical or topographic cues, the current understanding of how the mechanical environment influences migration remains largely understudied and incomplete. Therefore, the focus of this study was to evaluate Schwann cell migration and morphodynamics in the presence of stiffness gradients, which revealed that Schwann cells can follow extracellular gradients of increasing stiffness, in a form of directed migration termed durotaxis. METHODS: Polyacrylamide substrates were fabricated to mimic the range of stiffness found in peripheral nerve tissue. We assessed Schwann cell response to substrates that were either mechanically uniform or embedded with a shallow or steep stiffness gradient, respectively corresponding to the mechanical niche present during either the fluid phase or subsequent matrix phase of the peripheral nerve regeneration process. We examined cell migration (velocity and directionality) and morphology (elongation, spread area, nuclear aspect ratio, and cell process dynamics). We also characterized the surface morphology of Schwann cells by scanning electron microscopy. RESULTS: On laminin-coated polyacrylamide substrates embedded with either a shallow (∼0.04 kPa/mm) or steep (∼0.95 kPa/mm) stiffness gradient, Schwann cells displayed durotaxis, increasing both their speed and directionality along the gradient materials, fabricated with elastic moduli in the range found in peripheral nerve tissue. Uniquely and unlike cell behavior reported in other cell types, the durotactic response of Schwann cells was not dependent upon the slope of the gradient. When we examined whether durotaxis behavior was accompanied by a pro-regenerative Schwann cell phenotype, we observed altered cell morphology, including increases in spread area and the number, elongation, and branching of the cellular processes, on the steep but not the shallow gradient materials. This phenotype emerged within hours of the cells adhering to the materials and was sustained throughout the 24 hour duration of the experiment. Control experiments also showed that unlike most adherent cells, Schwann cells did not alter their morphology in response to uniform substrates of different stiffnesses. CONCLUSION: This study is notable in its report of durotaxis of cells in response to a stiffness gradient slope, which is greater than an order of magnitude less than reported elsewhere in the literature, suggesting Schwann cells are highly sensitive detectors of mechanical heterogeneity. Altogether, this work identifies durotaxis as a new migratory modality in Schwann cells, and further shows that the presence of a steep stiffness gradient can support a pro-regenerative cell morphology.

A three-dimensional neural spheroid model for capillary-like network formation.

BACKGROUND: In vitro three-dimensional neural spheroid models have an in vivo-like cell density, and have the potential to reduce animal usage and increase experimental throughput. The aim of this study was to establish a spheroid model to study the formation of capillary-like networks in a three-dimensional environment that incorporates both neuronal and glial cell types, and does not require exogenous vasculogenic growth factors. NEW METHOD: We created self-assembled, scaffold-free cellular spheroids using primary-derived postnatal rodent cortex as a cell source. The interactions between relevant neural cell types, basement membrane proteins, and endothelial cells were characterized by immunohistochemistry. Transmission electron microscopy was used to determine if endothelial network structures had lumens. RESULTS: Endothelial cells within cortical spheroids assembled into capillary-like networks with lumens. Networks were surrounded by basement membrane proteins, including laminin, fibronectin and collagen IV, as well as key neurovascular cell types. COMPARISON WITH EXISTING METHOD(S): Existing in vitro models of the cortical neurovascular environment study monolayers of endothelial cells, either on transwell inserts or coating cellular spheroids. These models are not well suited to study vasculogenesis, a process hallmarked by endothelial cell cord formation and subsequent lumenization. CONCLUSIONS: The neural spheroid is a new model to study the formation of endothelial cell capillary-like structures in vitro within a high cell density three-dimensional environment that contains both neuronal and glial populations. This model can be applied to investigate vascular assembly in healthy or disease states, such as stroke, traumatic brain injury, or neurodegenerative disorders.

Genomic and morphological changes of neuroblastoma cells in response to three-dimensional matrices.

Advances in neural tissue engineering require a comprehensive understanding of neuronal growth in 3 dimensions. This study compared the gene expression of SH-SY5Y human neuroblastoma cells cultured in 3-dimensional (3D) with those cultured in 2-dimensional (2D) environments. Microarray analysis demonstrated that, in response to varying matrix geometry, SH-SY5Y cells exhibited differential expression of 1,766 genes in collagen I, including those relevant to cytoskeleton, extracellular matrix, and neurite outgrowth. Cells extended longer neurites in 3D collagen I cultures than in 2D. Real-time reverse transcriptase polymerase chain reaction experiments and morphological analysis comparing collagen I and Matrigel tested whether the differential growth and gene expression reflected influences of culture dimension or culture material. SH-SY5Y neuroblastoma cells responded to geometry by differentially regulating cell spreading and genes associated with actin in similar patterns for both materials; however, neurite outgrowth and the expression of the gene encoding for neurofilament varied with the type of material. Electron microscopy and mechanical analysis showed that collagen I was more fibrillar than Matrigel, with larger inter-fiber distance and higher stiffness. Taken together, these results suggest complex cell-material interactions, in which the dimension of the culture material influences gene expression and cell spreading and the structural and mechanical properties of the culture material influence gene expression and neurite outgrowth.

Neurite bridging across micropatterned grooves.

After injury, regenerating axons must navigate complex, three-dimensional (3D) microenvironments. Topographic guidance of neurite outgrowth has been demonstrated in vitro with culture substrates that contain micropatterned features on the nanometer-micron scale. In this study we report the ability of microfabricated biomaterials to support neurite extension across micropatterned grooves with feature sizes on the order of tens of microns, sizes relevant to the design of biomaterials and tissue engineering scaffolds. Neonatal rat dorsal root ganglion (DRG) neurons were cultured on grooved substrates of poly(dimethyl siloxane) coated with poly-L-lysine and laminin. Here we describe an unusual capability of a subpopulation of DRG neurons to extend neurites that spanned across the grooves, with no underlying solid support. Multiple parameters influenced the formation of bridging neurites, with the highest numbers of bridges observed under the following experimental conditions: cell density of 125,000 cells per sample, groove depth of 50 microm, groove width of 30 microm, and plateau width of 200 microm. Bridges were formed as neurites extended from a neuron in a groove, contacted adjacent plateaus, pulled the neuron up to become suspended over the groove, and the soma translocated to the plateau. These studies are of interest to understanding cytoskeletal dynamics and designing biomaterials for 3D axon guidance.

Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations.

Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular ß-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.

A biomimetic synthetic feeder layer supports the proliferation and self-renewal of mouse embryonic stem cells.

UNLABELLED: Successful realization of the enormous potential of pluripotent stem cells in regenerative medicine demands the development of well-defined culture conditions. Maintenance of embryonic stem cells (ESCs) typically requires co-culture with feeder layer cells, generally mouse embryonic fibroblasts (MEFs). Concerns about xenogeneic pathogen contamination and immune reaction to feeder cells underlie the need for ensuring the safety and efficacy of future stem cell-based products through the development of a controlled culture environment. To gain insight into the effectiveness of MEF layers, here we have developed a biomimetic synthetic feeder layer (BSFL) that is acellular and replicates the stiffness and topography of MEFs. The mechanical properties of MEFs were measured using atomic force microscopy. The average Young's modulus of the MEF monolayers was replicated using tunable polyacrylamide (PA) gels. BSFLs replicated topographical features of the MEFs, including cellular, subcellular, and cytoskeletal features. On BSFLs, mouse ESCs adhered and formed compact round colonies; similar to on MEF controls but not on Flat PA. ESCs on BSFLs maintained their pluripotency and self-renewal across passages, formed embryoid bodies and differentiated into progenitors of the three germ layers. This acellular biomimetic synthetic feeder layer supports stem cell culture without requiring co-culture of live xenogeneic feeder cells, and provides a versatile, tailorable platform for investigating stem cell growth. STATEMENT OF SIGNIFICANCE: Embryonic stem cells have enormous potential to aid therapeutics, because they can renew themselves and become different cell types. This study addresses a key challenge for ESC use - growing them safely for human patients. ESCs typically grow with a feeder layer of mouse fibroblasts. Since patients have a risk of immune response to feeder layer cells, we have developed a material to mimic the feeder layer and eliminate this risk. We investigated the influence of feeder layer topography and stiffness on mouse ESCs. While the biomimetic synthetic feeder layer contains no live cells, it replicates the stiffness and topography of feeder layer cells. Significantly, ESCs grown on BSFLs retain their abilities to grow and become multiple cell types.

Comparison of three myofibroblast cell sources for the tissue engineering of cardiac valves.

The objective of this study was to evaluate the capacity of three clinically useful tissue sources: tricuspid valve leaflet (TVL), carotid artery (CA), and jugular vein (JV), to generate myofibroblasts for potential use in a tissue-engineered cardiac valve replacement. Tissue biopsies of clinically appropriate sizes obtained from juvenile sheep were used for this work. Cells obtained from all three tissue sources exhibited a myofibroblast phenotype in vitro, as demonstrated by their immunoreactivity with antibodies directed against vimentin, alpha-smooth muscle actin, fibronectin, and chondroitin sulfate. Protein synthesis characteristics were defined for the key extracellular matrix components: collagen, glycosaminoglycans, and elastin. Among the three sources, JV generated the highest numbers of cells, and JV cells produced the largest amount of collagen per cell. These data suggest that venous tissue, with its relative ease of accessibility, may generate myofibroblasts potentially useful for the interstitial cellular component of a tissue-engineered cardiac valve.

Application and assessment of optical clearing methods for imaging of tissue-engineered neural stem cell spheres.

Three-dimensional (3D) cell culture is an important tool that facilitates biological discoveries by bridging the divide between standard two-dimensional cell culture and the complex, high-cell-density in vivo environment. Typically, the internal structures of 3D tissue-engineered samples are visualized through an involved process of physical sectioning, immunostaining, imaging, and computational reconstruction. However, recent progress in tissue-clearing methods has improved optical-imaging-depth capabilities in whole embryos and brains by reducing tissue opacity and light scattering, thus decreasing the need for physical sectioning. In this study, we assessed the application of the recently published clearing techniques Clear(T2), Scale, and SeeDB to tissue-engineered neural spheres. We found that scaffold-free self-assembled adult hippocampal neural stem cell spheres of 100-µm diameter could be optically cleared and imaged using either Clear(T2) or Scale, while SeeDB only marginally improved imaging depth. The Clear(T2) protocol maintained sphere size, while Scale led to sample expansion, and SeeDB led to sample shrinkage. Additionally, using Clear(T2) we cleared and successfully imaged spheres of C6 glioma cells and spheres of primary cortical neurons. We conclude that Clear(T2) is the most effective protocol of those tested at clearing neural spheres of various cell types and could be applied to better understand neural cell interactions in 3D tissue-engineered samples.

Three-Dimensional Neural Spheroid Culture: An In Vitro Model for Cortical Studies.

There is a high demand for in vitro models of the central nervous system (CNS) to study neurological disorders, injuries, toxicity, and drug efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional two-dimensional culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry through both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the CNS in vitro.

Tricuspid valve biopsy: a potential source of cardiac myofibroblast cells for tissue-engineered cardiac valves.

BACKGROUND AND AIMS OF THE STUDY: As progress is made in the development of a tissue-engineered cardiac valve, the need for a reliable cell source is particularly important. A technique has been developed for the reliable biopsy of tricuspid valve leaflets. Expanding the harvested cells in culture is feasible and provides a source of leaflet cells that are structurally and functionally similar to the pulmonary and aortic valve leaflet cells that they may replace. METHODS: Thirteen sheep underwent tricuspid valve biopsy. Transthoracic echocardiography (TTE) was performed to evaluate function and guide the subsequent biopsy. Myofibroblasts were isolated from the biopsy samples, expanded in culture through 10 passages, and evaluated with immunocytochemistry for valve cell markers. Two animals were sacrificed acutely, two animals died during the immediate postoperative period, and nine animals survived for four weeks or more. RESULTS: All preoperative and pre-explantation echocardiograms were normal. Both animals sacrificed acutely showed that the tricuspid valve leaflet was indeed biopsied with this technique. Two perioperative deaths occurred; one animal died secondary to injury of the chorda tendinea with subsequent destruction of the posterior leaflet; another died from disruption of the superior vena cava that led to irreversible cardiac tamponade. At sacrifice (2 to 17 weeks), all other animals showed intact tricuspid valves with normal leaflet anatomy. All cultured biopsies generated myofibroblasts that were immunocytochemically positive for alpha smooth muscle actin, chondroitin sulfate, vimentin and fibronectin. CONCLUSION: Biopsy of the tricuspid valve to obtain recipient cardiac valve leaflet cells is possible, and the technique is simple and reliable. Biopsy of the leaflet does not compromise function. Interstitial cells can be harvested and expanded in culture. Cellular structure and function is preserved and is similar to that of other cardiac leaflet cells. Tricuspid valve leaflet biopsies are a potential source for harvesting cells to be used in the development of a tissue-engineered cardiac valve.

Three-dimensional traction forces of Schwann cells on compliant substrates.

The mechanical interaction between Schwann cells (SCs) and their microenvironment is crucial for the development, maintenance and repair of the peripheral nervous system. In this paper, we present a detailed investigation on the mechanosensitivity of SCs across a physiologically relevant substrate stiffness range. Contrary to many other cell types, we find that the SC spreading area and cytoskeletal actin architecture were relatively insensitive to substrate stiffness with pronounced stress fibre formation across all moduli tested (0.24-4.80 kPa). Consistent with the presence of stress fibres, we found that SCs generated large surface tractions on stiff substrates and large, finite material deformations on soft substrates. When quantifying the three-dimensional characteristics of the SC traction profiles, we observed a significant contribution from the out-of-plane traction component, locally giving rise to rotational moments similar to those observed in mesenchymal embryonic fibroblasts. Taken together, these measurements provide the first set of quantitative biophysical metrics of how SCs interact with their physical microenvironment, which are anticipated to aid in the development of tissue engineering scaffolds designed to promote functional integration of SCs into post-injury in vivo environments.