Understanding mechanobiology in cultured endothelium: A review of the orbital shaker method

Abstract

A striking feature of atherosclerosis is its highly non-uniform distribution within the arterial tree. This has been attributed to variation in the haemodynamic wall shear stress (WSS) experienced by endothelial cells, but the WSS characteristics that are important and the mechanisms by which they lead to disease remain subjects of intensive investigation despite decades of research. In vivo evidence suggests that multidirectional WSS is highly atherogenic. This possibility is increasingly being studied by culturing endothelial cells in wells that are swirled on an orbital shaker. The method is simple and cost effective, has high throughput and permits chronic exposure, but interpretation of the results can be difficult because the fluid mechanics are complex; hitherto, their description has largely been restricted to the engineering literature. Here we review the findings of such studies, which indicate that putatively atherogenic flow characteristics occur at the centre of the well whilst atheroprotective ones occur towards the edge, and we describe simple mathematical methods for choosing experimental variables that avoid resonance, wave breaking and uncovering of the cells. We additionally summarise a large number of studies showing that endothelium cultured at the centre of the well expresses more pro-inflammatory and fewer homeostatic genes, has higher permeability, proliferation, apoptosis and senescence, and shows more endothelial-to-mesenchymal transition than endothelium at the edge. This simple method, when correctly interpreted, has the potential to greatly increase our understanding of the homeostatic and pathogenic mechanobiology of endothelial cells and may help identify new therapeutic targets in vascular disease.

Endothelial reprogramming by disturbed flow revealed by single-cell RNA and chromatin accessibility study

“…Consistent with the accessibility changes, the corresponding gene expression levels were also increased in ECs exposed to d-flow in E8. These results confirm that d-flow indeed induced EndMT as expected (Glaser et al, 2020;Lai et al, 2018;Mahmoud et al, 2017;Moonen et al, 2015;Ten Dijke et al, 2012;Warboys et al, 2019).…”

Computational Characterization of the Dish-In-A-Dish, A High Yield Culture Platform for Endothelial Shear Stress Studies on the Orbital Shaker

“…The flow characteristics that define atheroprone and atheroprotective regimes are complex, but it has been established that low τ max and OSI are atheroprone and correlate with atherogenesis [19,35]. We aimed for dishes with a high τ max and a low OSI, while maximizing the cell culture area in the dish.…”

“…As a result, a non-uniform shear stress field is created that depends on the angular velocity and the radius of the shaker. Although this approach increases the experimental throughput significantly, the distribution of the shear stress is inhomogeneous and includes both atheroprone and atheroprotective regions [18,19]. These conflicting mechanical cues lead to conflicting biological responses.…”

“…The resulting shear stress values are based on the orbit size of the orbital shaker and the angular velocity. This analytical approach assumes unperturbed fluid in the far field, neglecting the side walls and initial fluid height [19,25,26]. Computational fluid dynamics (CFD) has been shown to be a more precise method to characterize flow and shear stress patterns in dishes on the orbital shaker [18,[27][28][29].…”

Integrating Biophysics in Toxicology

“…Following these strategies, there are examples for the modulation of substrate stiffness [77] or shear stress [140]. An additional possibility includes the use of rather common laboratory equipment like in the orbital-shaker method for the cultivation of plates under shear stress [173]. Moreover, commercial systems are more and more diffuse and offer support and standardization of the experimental conditions [174].…”