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.

Human-induced pluripotent stem cell-derived cardiomyocytes are a potentially unlimited cell source and promising patient-specific in vitro model of cardiac diseases. Yet, these cells are limited by immaturity and population heterogeneity. Current in vitro studies aiming at better understanding of the mechanical and chemical cues in the microenvironment that drive cellular maturation involve deformable materials and precise manipulation of the microenvironment with, for example, micropatterns. Such microenvironment manipulation most often involves microfabrication protocols which are time-consuming, require cleanroom facilities and photolithography expertise. Here, we present a method to increase the scale of the fabrication pipeline, thereby enabling large-batch generation of shelf-stable microenvironment protein templates on glass chips. This decreases fabrication time and allows for more flexibility in the subsequent steps, for example, in tuning the material properties and the selection of extracellular matrix or cell proteins. Further, the fabrication of deformable hydrogels has been optimized for compatibility with these templates, in addition to the templates being able to be used to acquire protein patterns directly on the glass chips. With our approach, we have successfully controlled the shapes of cardiomyocytes seeded on Matrigel-patterned hydrogels.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in-vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. To understand how cellular mechanisms affect the heart, it is crucial to quantify the contractile function in single hiPSC-CMs over time, however, such measurements remain demanding and low-throughput, and are too seldom considered. We developed an open-access, versatile, streamlined, and highly automated pipeline to address these challenges and enable quantitative tracking of the contractile dynamics of single hiPSC- CMs over time: ConTraX. Three interlocking software modules enable: (i) parameter-based localization and selection of single hiPSC-CMs; (ii) automated video acquisition of >200 cells/hour; and (iii) streamlined measurements of the contractile parameters via traction force microscopy. Using ConTraX, we analyzed >2,753 hiPSC-CMs over time under orthogonal experimental conditions in terms of culture media and substrate stiffnesses. Using undirected high-dimensional clustering, we dissected the complex diversity of contractile phenotypes in hiPSC-CM populations and revealed converging maturation patterns. Our modular ConTraX pipeline empowers biologists with a potent quantitative analytic tool applicable to the development of cardiac therapies. ### Competing Interest Statement The authors have declared no competing interest. * hiPSC : Human-induced pluripotent stem cell CM : Cardiomyocyte TFM : Traction force microscopy PIV : Particle image velocimetry DIC : Digital image correlation FTTC : Fourier transform traction cytometry GUI : Graphical User Interface

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.

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.

The ability to differentiate human cardiomyocytes (heart muscle cells) from induced pluripotent stem cells (iPSCs) presents high potential to model heart contractility. Shortening of sarcomeres in series along intracellular myofibrils enables the beating of cardiomyocytes. We developed a platform with patterned single iPSC-cardiomyocytes in arrays and tested the contractility of these cells as a function of their shape and of substrate stiffness. We fluorescently labelled sarcomeres with LifeAct to analyze their shortening and organization. We measured the mechanical output of contractile cycles with traction force microscopy and adapted cross-correlation algorithms to characterize movement of sarcomeres. We assayed single cells on patterns of 2000 μm2 with aspect ratios (length:width) ranging from 1:1 to 7:1 and on substrates with varied stiffnesses: 6 kPa, 10 kPa and 35 kPa. Preliminary studies suggested that cell morphology and substrate stiffness affect the organization of sarcomeres with a potential impact in cardiomyocyte contractility. We aimed to understand how the contractility of iPSC-cardiomyocytes is affected by the improved organization of sarcomeres induced by these cues. For a substrate stiffness of 10 kPa, we observed that cells with a 7:1 aspect ratio produced higher contractile forces per μm of sarcomere shortening. Improved sarcomere alignment seemed to drive increased contractility of cells with this shape. Substrate stiffness also affected the contractility of 7:1 iPSC-cardiomyocytes. 35 kPa substrates induced sarcomere ruptures and lower contractile forces. Cells on 6 kPa substrates developed sarcomeres that buckled. These results suggested a role of intracellular tension in contractility. We further tested how tension affects contractility and sarcomere organization in 7:1 cells through cell stretching, culture in high calcium and by inhibiting non-muscle myosin. Results showed evidence that a tuned intracellular tension mechanism drives myofibril alignment and improved contractility in iPSC-cardiomyocytes.

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.

Recovery from peripheral nerve damage, especially for a transected nerve, is rarely complete, resulting in impaired motor function, sensory loss, and chronic pain with inappropriate autonomic responses that seriously impair quality of life. In consequence, strategies for enhancing peripheral nerve repair are of high clinical importance. Tension is a key determinant of neuronal growth and function. In vitro and in vivo experiments have shown that moderate levels of imposed tension (strain) can encourage axonal outgrowth; however, few strategies of peripheral nerve repair emphasize the mechanical environment of the injured nerve. Toward the development of more effective nerve regeneration strategies, we demonstrate the design, fabrication, and implementation of a novel, modular nerve-lengthening device, which allows the imposition of moderate tensile loads in parallel with existing scaffold-based tissue engineering strategies for nerve repair. This concept would enable nerve regeneration in two superposed regimes of nerve extension--traditional extension through axonal outgrowth into a scaffold and extension in intact regions of the proximal nerve, such as that occurring during growth or limb-lengthening. Self-sizing silicone nerve cuffs were fabricated to grip nerve stumps without slippage, and nerves were deformed by actuating a telescoping internal fixator. Poly(lactic co-glycolic) acid (PLGA) constructs mounted on the telescoping rods were apposed to the nerve stumps to guide axonal outgrowth. Neuronal cells were exposed to PLGA using direct contact and extract methods, and they exhibited no signs of cytotoxic effects in terms of cell morphology and viability. We confirmed the feasibility of implanting and actuating our device within a sciatic nerve gap and observed axonal outgrowth following device implantation. The successful fabrication and implementation of our device provides a novel method for examining mechanical influences on nerve regeneration.

: Peripheral nerve damage is one consequence of injury to the extremities of soldiers by improvised explosive devices (IEDs). The degree of functional recovery from peripheral nerve damage is often poor, particularly for severed nerves. The result can be impaired motor function, sensory loss, and chronic pain with inappropriate autonomic responses. Consequently, strategies for enhancing nervous function are of high military relevance. Towards the development of more effective nerve regeneration strategies, this proposal addresses the hypothesis that moderate tensile loading (stretch) of peripheral nerves can stabilize nerve degradation and also promote accelerated regeneration. Our project aims are to 1) To examine the impact of low levels of tensile loading on the Wallerian degeneration of proximal and distal stumps of severed peripheral nerves and 2) To examine the impact of moderate levels of tensile loading on promoting the outgrowth and functional connectivity of severed peripheral nerves. To meet these aims, in the second project period, based on results from initial in vivo implantations, we modified our two nerve lengthening devices, one a fixed length and one extensible, and are currently performing long-term implantations to test the effectiveness of these devices for nerve regeneration.

The advent of microbubble contrast agents has enhanced the capabilities of ultrasound as a medical imaging modality and stimulated innovative strategies for ultrasound-mediated drug and gene delivery. While the utilization of microbubbles as carrier vehicles has shown encouraging results in cancer therapy, their applicability has been limited by a large size which typically confines them to the vasculature. To enhance their multifunctional contrast and delivery capacity, it is critical to reduce bubble size to the nanometer range without reducing echogenicity. In this work, we present a novel strategy for formulation of nanosized, echogenic lipid bubbles by incorporating the surfactant Pluronic, a triblock copolymer of ethylene oxide copropylene oxide coethylene oxide into the formulation. Five Pluronics (L31, L61, L81, L64 and P85) with a range of molecular weights (M-w: 1100 to 4600 Da) were incorporated into the lipid shell either before or after lipid film hydration and before addition of perfluorocarbon gas. Results demonstrate that Pluronic-lipid interactions lead to a significantly reduced bubble size. Among the tested formulations, bubbles made with Pluronic L61 were the smallest with a mean hydrodynamic diameter of 207.9 +/- 74.7 nm compared to the 880.9 +/- 127.6 nm control bubbles. Pluronic L81 also significantly reduced bubble size to 406.8 +/- 21.0 nm. We conclude that Pluronic is effective in lipid bubble size control, and Pluronic M-w, hydrophilic-lipophilic balance (HLB), and Pluronic/lipid ratio are critical determinants of the bubble size. Most importantly, our results have shown that although the bubbles are nanosized, their stability and in vitro and in vivo echogenicity are not compromised. The resulting nanobubbles may be better suited for contrast enhanced tumor imaging and subsequent therapeutic delivery.