Toward a Physiologically Relevant 3D Helicoidal-Oriented Cardiac Model: Simultaneous Application of Mechanical Stimulation and Surface Topography.

Myocardium consists of cardiac cells that interact with their environment through physical, biochemical, and electrical stimulations. The physiology, function, and metabolism of cardiac tissue are affected by this dynamic structure. Within the myocardium, cardiomyocytes' orientations are parallel, creating a dominant orientation. Additionally, local alignments of fibers, along with a helical organization, become evident at the macroscopic level. For the successful development of a reliable in vitro cardiac model, evaluation of cardiac cells' behavior in a dynamic microenvironment, as well as their spatial architecture, is mandatory. In this study, we hypothesize that complex interactions between long-term contraction boundary conditions and cyclic mechanical stimulation may provide a physiological mechanism to generate off-axis alignments in the preferred mechanical stretch direction. This off-axis alignment can be engineered in vitro and, most importantly, mirrors the helical arrangements observed in vivo. For this purpose, uniaxial mechanical stretching of dECM-fibrin hydrogels was performed on pre-aligned 3D cultures of cardiac cells. In view of the potential development of helical structures similar to those in native hearts, the possibility of generating oblique alignments ranging between 0° and 90° was explored. Indeed, our investigations of cell alignment in 3D, employing both mechanical stimulation and groove constraint, provide a reliable mechanism for the generation of helicoidal structures in the myocardium. By combining cyclic stretch and geometric alignment in grooves, an intermediate angle toward favored direction can be achieved experimentally: while cyclic stretch produces a perpendicular orientation, geometric alignment is associated with a parallel one. In our 2D and 3D culture conditions, nonlinear cellular addition of the strains and strain avoidance concept reliably predicted the preferred cellular alignment. The 3D dECM-fibrin model system in this study shows that cyclical stretching supports cell survival and development. Using mechanical stimulation of pre-aligned heart cells, maturation markers are augmented in neonatal cardiomyocytes, while the beating culture period is prolonged, indicating an improved model function. We propose a simplified theoretical model based on numerical simulation and nonlinear strain avoidance by cells to explain oblique alignment angles. Thus, this work lays a possible rational basis for understanding and engineering oblique cellular alignments, such as the helicoidal layout of the heart, using approaches that simultaneously enhance maturation and function.

Multimaterial Nanoporous Membranes Shaped through High Aspect-Ratio Sacrificial Silicon Nanostructures.

We present an innovative fabrication method for solid-state nanoporous membranes based on the casting of sacrificial silicon nanostructures. The process allows the individual definition of geometry and placement of each nanopore through e-beam lithography and is compatible with a wide range of materials without the need to adapt the process to the materials used. We demonstrate the fabrication of membranes integrating high aspect-ratio nanopores with critical dimensions as small as 30 nm, 1.2 µm in length, with round or elongated shapes, and made of silicon dioxide or amorphous carbon. The capability to engineer nanoporous membranes made of a variety of materials and with tailored designs will lead to new applications in the field of electrochemical sensing, flow modulation, or the chemical functionalization of nanopores.

Ultra-soft cantilevers and 3-D micro-patterned substrates for contractile bundle tension measurement in living cells.

Actin-myosin microfilament bundles or stress-fibers are the principal tension-generating structures in the cell. Their mechanical properties are critical for cell shape, motion, and interaction with other cells and extracellular matrix, but were so far difficult to access in a living cell. Here we propose a micro-fabricated two-component setup for direct tension measurement on a peripheral bundle within an intact cell. We used 3-D substrates made of silicon elastomer to elevate the cell making the filament bundle at its border accessible from the side, and employed an ultra-soft (spring constant 0.78 nN µm(-1)) epoxy-based cantilever for mechanical probing. With this setup we were able for the first time to measure the tension in peripheral actin bundles in living primary fibroblasts spread on a rigid substrate.

Contact angle at the leading edge controls cell protrusion rate.

Plasma membrane tension and the pressure generated by actin polymerization are two antagonistic forces believed to define the protrusion rate at the leading edge of migrating cells [1-5]. Quantitatively, resistance to actin protrusion is a product of membrane tension and mean local curvature (Laplace's law); thus, it depends on the local geometry of the membrane interface. However, the role of the geometry of the leading edge in protrusion control has not been yet investigated. Here, we manipulate both the cell shape and substrate topography in the model system of persistently migrating fish epidermal keratocytes. We find that the protrusion rate does not correlate with membrane tension, but, instead, strongly correlates with cell roundness, and that the leading edge of the cell exhibits pinning on substrate ridges-a phenomenon characteristic of spreading of liquid drops. These results indicate that the leading edge could be considered a triple interface between the substrate, membrane, and extracellular medium and that the contact angle between the membrane and the substrate determines the load on actin polymerization and, therefore, the protrusion rate. Our findings thus illuminate a novel relationship between the 3D shape of the cell and its dynamics, which may have implications for cell migration in 3D environments.

Separation of platelets from other blood cells in continuous-flow by dielectrophoresis field-flow-fractionation.

We present a microfluidic device capable of separating platelets from other blood cells in continuous flow using dielectrophoresis field-flow-fractionation. The use of hydrodynamic focusing in combination with the application of a dielectrophoretic force allows the separation of platelets from red blood cells due to their size difference. The theoretical cell trajectory has been calculated by numerical simulations of the electrical field and flow speed, and is in agreement with the experimental results. The proposed device uses the so-called "liquid electrodes" design and can be used with low applied voltages, as low as 10 V(pp). The obtained separation is very efficient, the device being able to achieve a very high purity of platelets of 98.8% with less than 2% cell loss. Its low-voltage operation makes it particularly suitable for point-of-care applications. It could further be used for the separation of other cell types based on their size difference, as well as in combination with other sorting techniques to separate multiple cell populations from each other.

Continuous-flow electrical lysis device with integrated control by dielectrophoretic cell sorting.

We present a device capable of electrical cell lysis and evaluation of lysis efficiency in continuous flow using dielectrophoretic cell sorting. We use a combination of AC electrical fields and so-called liquid electrodes to avoid bubble creation at the electrode surface. The electrical field distribution is calculated in different electrode configurations by numerical simulations. Cell sorting shows high lysis efficiency, 99% of yeast cells sorted after lysis featuring dielectric properties similar to dead cells. A study of the potential device throughput is performed.

Blood cell counting by means of impedance measurements in a microsystem device.

This paper presents an innovative, portable and low power blood cell counter, based on micro-electro-mechanical-systems (MEMS) technology. It was realized by designing and developing a custom impedance measurement circuit, which drives an electro-fluidic microsystem, providing a parallel, multi-channel Coulter counter. A method for a reliable, easy, and low-cost interfacing to such kind of micro-devices, allowing both fluidic and electric coupling, is also shown. Preliminary experiments led to promising results: fluidics works properly without leakages or clogging. Electrodes show good stability with current (in terms of adhesion), and measured channel impedances are satisfyingly low (30 kW for a cubic Coulter orifice, side 10 microm). Finally, we present a possible extension of the setup, based on a dual-characteristic, electro-optical counting.