Studying the Synergistic Effect of Substrate Stiffness and Cyclic Stretch Level on Endothelial Cells Using an Elastomeric Cell Culture Chamber.

Endothelial cells lining blood vessels are continuously exposed to biophysical cues that regulate their function in health and disease. As we age, blood vessels lose their elasticity and become stiffer. Vessel stiffness alters the mechanical forces that endothelial cells experience. Despite ample evidence on the contribution of endothelial cells to vessel stiffness, less is known about how vessel stiffness affects endothelial cells. In this study, we developed a versatile model to study the cooperative effect of substrate stiffness and cyclic stretch on human aortic endothelial cells. We cultured endothelial cells on elastomeric wells covered with fibronectin-coated polyacrylamide gel. Varying the concentrations of acrylamide and bis-acrylamide enabled us to produce soft and stiff substrates with elastic modules of 40 and 200 kPa, respectively. Using a customized three-dimensional (3D) printed cam-driven system, the cells were exposed to 5 and 10% cyclic stretch levels. This enabled us to mimic the stiffness and stretch levels that endothelial cells experience in young and aged arteries. Using this model, we found that endothelial cells cultured on a soft substrate had minimal cytoskeletal alignment to the direction of the stretch compared to the ones cultured on the stiff substrate. We also observed an increase in the cellular area and aspect ratio in cells cultured on the stiff substrate, both of which are positively regulated by cyclic stretch. However, neither cyclic stretch nor substrate stiffness significantly affected the nuclear circularity. Additionally, we found that the accumulation of NF-κB in the nucleus, endothelial proliferation, tube formation, and expression of IL1ß depends on the stretch level and substrate stiffness. Our model can be further used to investigate the complex signaling pathways associated with vessel stiffening that govern the endothelial responses to mechanical forces.

Studying the Mechanobiology of Aortic Endothelial Cells Under Cyclic Stretch Using a Modular 3D Printed System.

Here, we describe a motorized cam-driven system for the cyclic stretch of aortic endothelial cells. Our modular design allows for generating customized spatiotemporal stretch profiles by varying the profile and size of 3D printed cam and follower elements. The system is controllable, compact, inexpensive, and amenable for parallelization and long-term experiments. Experiments using human aortic endothelial cells show significant changes in the cytoskeletal structure and morphology of cells following exposure to 5 and 10% cyclic stretch over 9 and 16 h. The system provides upportunities for exploring the complex molecular and cellular processes governing the response of mechanosensitive cells under cyclic stretch.