Force spectroscopy has been used to measure the adhesion of Saos-2 cells to a glass surface at different phases of the cell cycle. The cells were synchronized in three phases of the cell cycle: G1, S, and G2M. Cells in these phases were compared with unsynchronized and native mitotic cells. Individual cells were attached to an atomic force microscope cantilever, brought into brief contact with the glass surface, and then pulled off again. The force-distance curves obtained allowed the work and maximum force of detachment as well as the number, amplitude, and position of discrete unbinding steps to be determined. A statistical analysis of the data showed that the number of binding proteins or protein complexes present at the cell surface and their binding properties remain similar throughout the cell cycle. This, despite the huge changes in cell morphology and adhesion that occur as the cells enter mitosis. These changes are rather associated with the changes in cytoskeletal organization, which can be quantified by force spectroscopy as changes in cell stiffness.

Cardiomyocyte alignment in myocardium tissue plays a significant role in the physiological, electrical, and mechanical functions of the myocardium. It remains, however, difficult to align cardiac cells in a 3D in vitro heart model. This paper proposes a simple method to align cells using microfabricated Polydimethylsiloxane (PDMS) grooves with large dimensions (of up to 350 µm in width), similar to the dimensions of trabeculae carneae, the smallest functional unit of the myocardium. Two cell groups were used in this work; first, H9c2 cells in combination with Nor10 cells for proof of concept, and second, neonatal cardiac cells to investigate the functionality of the 3D model. This model compared the patterned and nonpatterned 3D constructs, as well as the 2D cell cultures, with and without patterns. In addition to alignment, we assessed the functionality of our proposed 3D model by comparing beating rates between aligned and non-aligned structures. In order to assess the practicality of the model, the 3D aligned structures should be demonstrated to be detachable and alignable. This evaluation is crucial to the use of this 3D functional model in future studies related to drug screening, building blocks for tissue engineering, and as a heart-on-chip by integrating microfluidics.

Complex 3D in vitro models are emerging as a key technology to support research areas in personalised medicine. Methods to manipulate and control the position of microtissues are needed to efficiently assemble many 3D in vitro models.

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.