Tracking single hiPSC-derived cardiomyocyte contractile function using CONTRAX an efficient pipeline for traction force measurement.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.

Field Guide to Traction Force Microscopy.

Introduction: Traction force microscopy (TFM) is a widely used technique to measure cell contractility on compliant substrates that mimic the stiffness of human tissues. For every step in a TFM workflow, users make choices which impact the quantitative results, yet many times the rationales and consequences for making these decisions are unclear. We have found few papers which show the complete experimental and mathematical steps of TFM, thus obfuscating the full effects of these decisions on the final output. Methods: Therefore, we present this "Field Guide" with the goal to explain the mathematical basis of common TFM methods to practitioners in an accessible way. We specifically focus on how errors propagate in TFM workflows given specific experimental design and analytical choices. Results: We cover important assumptions and considerations in TFM substrate manufacturing, substrate mechanical properties, imaging techniques, image processing methods, approaches and parameters used in calculating traction stress, and data-reporting strategies. Conclusions: By presenting a conceptual review and analysis of TFM-focused research articles published over the last two decades, we provide researchers in the field with a better understanding of their options to make more informed choices when creating TFM workflows depending on the type of cell being studied. With this review, we aim to empower experimentalists to quantify cell contractility with confidence. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00801-6.

3D Microwell Platforms for Control of Single Cell 3D Geometry and Intracellular Organization.

INTRODUCTION: Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization. METHODS: We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape. RESULTS: We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment. CONCLUSIONS: Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

Controlling cell shape on hydrogels using lift-off protein patterning.

Polyacrylamide gels functionalized with extracellular matrix proteins are commonly used as cell culture platforms to evaluate the combined effects of extracellular matrix composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a high-fidelity photoresist lift-off patterning method to pattern ECM proteins on polyacrylamide hydrogels with elastic moduli ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing method. Lift-off patterning achieves higher protein transfer efficiency, increases pattern accuracy, increases pattern yield, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape and enable long-term imaging of actin intracellular structure and lamellipodia dynamics when we culture epithelial cells on these substrates.

Changes in E-cadherin rigidity sensing regulate cell adhesion.

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

Tuning the Range of Polyacrylamide Gel Stiffness for Mechanobiology Applications.

Adjusting the acrylamide monomer and cross-linker content in polyacrylamide gels controls the hydrogel stiffness, yet the reported elastic modulus for the same formulations varies widely and these discrepancies are frequently attributed to different measurement methods. Few studies exist that examine stiffness trends across monomer and cross-linker concentrations using the same characterization platform. In this work, we use Atomic Force Microscopy and analyze force-distance curves to derive the elastic modulus of polyacrylamide hydrogels. We find that gel elastic modulus increases with increasing cross-link concentration until an inflection point, after which gel stiffness decreases with increasing cross-linking. This behavior arises because of the formation of highly cross-linked clusters, which add inhomogeneity and heterogeneity to the network structure, causing the global network to soften even under high cross-linking conditions. We identify these inflection points for three different total polymer formulations. When we alter gelation kinetics by using a low polymerization temperature, we find that gels are stiffer when polymerized at 4 °C compared to room temperature, indicating a complex relationship between gel structure, elasticity, and network formation. We also investigate how gel stiffness changes during storage over 10 days and find that specific gel formulations undergo significant stiffening (1.55 ± 0.13), which may be explained by differences in gel swelling resulting from initial polymerization parameters. Taken together, our study emphasizes the importance of polyacrylamide formulation, polymerization temperature, gelation time, and storage duration in defining the structural and mechanical properties of the polyacrylamide hydrogels.

For whom the cells pull: Hydrogel and micropost devices for measuring traction forces.

While performing several functions, adherent cells deform their surrounding substrate via stable adhesions that connect the intracellular cytoskeleton to the extracellular matrix. The traction forces that deform the substrate are studied in mechanotrasduction because they are affected by the mechanics of the extracellular milieu. We review the development and application of two methods widely used to measure traction forces generated by cells on 2D substrates: (i) traction force microscopy with polyacrylamide hydrogels and (ii) calculation of traction forces with arrays of deformable microposts. Measuring forces with these methods relies on measuring substrate displacements and converting them into forces. We describe approaches to determine force from displacements and elaborate on the necessary experimental conditions for this type of analysis. We emphasize device fabrication, mechanical calibration of substrates and covalent attachment of extracellular matrix proteins to substrates as key features in the design of experiments to measure cell traction forces with polyacrylamide hydrogels or microposts. We also report the challenges and achievements in integrating these methods with platforms for the mechanical stimulation of adherent cells. The approaches described here will enable new studies to understand cell mechanical outputs as a function of mechanical inputs and advance the understanding of mechanotransduction mechanisms.

A biotic game design project for integrated life science and engineering education.

Engaging, hands-on design experiences are key for formal and informal Science, Technology, Engineering, and Mathematics (STEM) education. Robotic and video game design challenges have been particularly effective in stimulating student interest, but equivalent experiences for the life sciences are not as developed. Here we present the concept of a "biotic game design project" to motivate student learning at the interface of life sciences and device engineering (as part of a cornerstone bioengineering devices course). We provide all course material and also present efforts in adapting the project's complexity to serve other time frames, age groups, learning focuses, and budgets. Students self-reported that they found the biotic game project fun and motivating, resulting in increased effort. Hence this type of design project could generate excitement and educational impact similar to robotics and video games.

Structural and molecular interrogation of intact biological systems.

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.

Post-collection processing of Schirmer strip-collected human tear fluid impacts protein content.

We examine the impact of post-collection sample handling on the protein composition of human tear samples. In particular, we characterize diffusion-based protein extraction from Schirmer strips. These strips of filter paper membrane are the de facto standard for tear fluid collection and storage, with diffusion-based protein elution off the strip being the most widely reported protein extraction strategy. Nevertheless, the diffusion-based protein elution strategy remains uncharacterized regarding downstream functional protein assays. Here, the time-dependence, concentration-dependence, and repeatability of the diffusion-based protein recovery protocol are characterized. Levels of protein irrecoverable from the Schirmer strip and lost during sample handling are isolated and compared for several major tear proteins. Further, the impact of the Schirmer strip and sample handling on the downstream concentration of proteins ranging in molecular weight, surface charge, and surface hydropathicity is quantified. Diffusion-based protein extraction from Schirmer strips was observed to be protein-dependent. Schirmer strips retained tear proteins to varying extents: 14.2% of lysozyme, 9.5% of human serum albumin, 27.7% of secretory IgA, and 30.9% of mucin 4. Tear protein loss during sample handling ranged from 2% (lysozyme) to 41.2% (mucin 4). Strip retention of protein was observed to be associated with protein molecular weight and hydrophobic surface area. Greater sample handling loss was associated with increased hydrophobic surface area of model proteins. Surface charge or surface hydrophilicity was not significantly associated with protein loss. We therefore conclude that, although diffusion-based processing of Schirmer strip-collected tear samples is widely used, these protocols may result in total post-collection protein loss which is considerable, consistent, and protein-dependent. This loss alters the relative and absolute protein concentrations in the sample. A priori prediction of strip-losses for individual proteins does not appear to be facile, based on cursory knowledge of protein surface properties. Thus, we emphasize "spike and recover" control experiments to determine expected elution profiles for target proteins when using diffusion-based protein sample preparation for Schirmer strip-collected tear fluid.