Single cardiomyocytes contain myofibrils that harbor the sarcomerebased contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetallike misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-μm 2 rectangles with length:width aspect ratios of 5:1-7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.contractility | sarcomeres | cardiomyocyte | stem cell | single cell

In this paper, we present a stretchable microelectrode array for studying cell behavior under mechanical strain. The electrode array consists of gold-plated nail-head pins (250 μm tip diameter) or tungsten micro-wires (25.4 μm in diameter) inserted into a polydimethylsiloxane (PDMS) platform (25.4 × 25.4 mm 2). Stretchable interconnects to the outside were provided by fusible indium-alloy-filled microchannels. The alloy is liquid at room temperature, thus providing the necessary stretchability and electrical conductivity. The electrode platform can withstand strains of up to 40% and repeated (100 times) strains of up to 35% did not cause any failure in the electrodes or the PDMS substrate. We confirmed biocompatibility of short-term culture, and using the gold pin device, we demonstrated electric field pacing of adult murine heart cells. Further, using the tungsten microelectrode device, we successfully measured depolarizations of differentiated murine heart cells from embryoid body clusters.

No abstract

No abstract

We have designed, fabricated, calibrated and tested actuators for shear characterization to assess microscale shear properties of soft substrates. Here we demonstrate characterization of dry silicone and hydrated polyethelyne glycol. Microscale tools, including atomic force microscopes and nanoindenters, often have limited functionality in hydrated environments. While electrostatic comb-drive actuators are particularly susceptible to moisture damage, through chemical vapor deposition of hexamethyldisiloxane, we increase the hydrophobicity of our electrostatic devices to a water contact angle 90 ± 3°. With this technique we determine the effective shear stiffness of both dry and hydrated samples for a range of soft substrates. Using computational and analytical models, we compare our empirically determined effective shear stiffness with existing characterization methods, rheology and nanoindentation, for samples with shear moduli ranging from 5-320 kPa. This work introduces a new approach for microscale assessment of synthetic materials that can be used on biological materials for basic and applied biomaterials research.