segmenting-growth-endothelial-cells-6-well-plates-on-an-orbital

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Imperial College London

Transport of macromolecules across vascular endothelium and its modification by fluid mechanical forces are important for normal tissue function and in the development of atherosclerosis. However, the routes by which macromolecules cross endothelium, the hemodynamic stresses that maintain endothelial physiology or trigger disease, and the dependence of transendothelial transport on hemodynamic stresses are controversial. We visualized pathways for macromolecule transport and determined the effect on these pathways of different types of flow. Endothelial monolayers were cultured under static conditions or on an orbital shaker producing different flow profiles in different parts of the wells. Fluorescent tracers that bound to the substrate after crossing the endothelium were used to identify transport pathways. Maps of tracer distribution were compared with numerical simulations of flow to determine effects of different shear stress metrics on permeability. Albumin-sized tracers dominantly crossed the cultured endothelium via junctions between neighboring cells, high-density lipoprotein-sized tracers crossed at tricellular junctions, and low-density lipoprotein-sized tracers crossed through cells. Cells aligned close to the angle that minimized shear stresses across their long axis. The rate of paracellular transport under flow correlated with the magnitude of these minimized transverse stresses, whereas transport across cells was uniformly reduced by all types of flow. These results contradict the long-standing two-pore theory of solute transport across microvessel walls and the consensus view that endothelial cells align with the mean shear vector. They suggest that endothelial cells minimize transverse shear, supporting its postulated proatherogenic role. Preliminary data show that similar tracer techniques are practicable in vivo. Solutes of increasing size crossed cultured endothelium through intercellular junctions, through tricellular junctions, or transcellularly. Cells aligned to minimize the shear stress acting across their long axis. Paracellular transport correlated with the level of this minimized shear, but transcellular transport was reduced uniformly by flow regardless of the shear profile.

Visualization of three pathways for macromolecule transport across cultured endothelium and their modification by flow.

Effects of shear stress on endothelium are important for the normal physiology of blood vessels and are implicated in the pathogenesis of atherosclerosis. They have been extensively studied in vitro. In one paradigm, endothelial cells are cultured in devices that produce spatially varying shear stress profiles, and the local profile is compared with the properties of cells at the same position. A flaw in this class of experiments is that cells exposed to a certain shear profile in one location may release mediators into the medium that alter the behaviour of cells at another location, experiencing different shear, thus obscuring or corrupting the true relation between shear and cell properties.

A novel method for segmenting growth of cells in sheared endothelial culture reveals the secretion of an anti-inflammatory mediator.

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.

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

Transport of water and solutes across vascular endothelium is important in normal physiology and critical in the development of various diseases, including atherosclerosis. However, there is debate about the routes for such transport. We recently showed that an albumin-sized tracer crossed endothelium at bicellular and tricellular junctions, a tracer having the size of high density lipoprotein crossed only through tricellular junctions, and a tracer with the size of low density lipoprotein was unable to cross by either route and instead traversed the cells themselves. Here we review previous work on the structure and function of tricellular junctions. We then describe a study in which we assessed the role of such junctions in the transport of an albumin-sized tracer.

The Role of Tricellular Junctions in the Transport of Macromolecules Across Endothelium.

The alignment of arterial endothelial cells (ECs) with the mean wall shear stress (WSS) vector is the prototypical example of their responsiveness to flow. However, evidence for this behaviour rests on experiments where many WSS metrics had the same orientation or where they were incompletely characterized. In the present study, we tested the phenomenon more rigorously. Aortic ECs were cultured in cylindrical wells on the platform of an orbital shaker. In this system, orientation would differ depending on the WSS metric to which the cells aligned. Variation in flow features and hence in possible orientations was further enhanced by altering the viscosity of the medium. Orientation of endothelial nuclei was compared with WSS characteristics obtained by computational fluid dynamics. At low mean WSS magnitudes, ECs aligned with the modal WSS vector, while at high mean WSS magnitudes they aligned so as to minimize the shear acting across their long axis (transverse WSS). Their failure to align with the mean WSS vector implies that other aspects of endothelial behaviour attributed to this metric require re-examination. The evolution of a mechanism for minimizing transverse WSS is consistent with it having detrimental effects on the cells and with its putative role in atherogenesis.

Article	Year
Visualization of three pathways for macromolecule transport across cultured endothelium and their modification by flow. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 28754719	2017
A novel method for segmenting growth of cells in sheared endothelial culture reveals the secretion of an anti-inflammatory mediator. Journal of biological engineering\| PubMed ID: 30127849	2018
Understanding mechanobiology in cultured endothelium: A review of the orbital shaker method. Atherosclerosis\| PubMed ID: 31096159	2019
The Role of Tricellular Junctions in the Transport of Macromolecules Across Endothelium. Cardiovascular engineering and technology\| PubMed ID: 32820467	2021
Endothelial cells do not align with the mean wall shear stress vector. Journal of the Royal Society, Interface\| PubMed ID: 33435845	2021

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