The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Summary

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Abstract

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Introduction

Fluid shear stress has been shown to modulate endothelial gene programs^1-5 through activation of cis-regulatory elements⁶, histone acetyltransferase activity⁷ and shear stress response elements (SSRE)⁸. Shear stress influences endothelial contributions towards coagulation by modulating tissue factor⁹ and tissue plasminogen activator (tPA)¹⁰ expression. Shear stress also influences control of angiogenesis¹¹ and vessel remodeling by regulating PDGF-B synthesis and responsiveness⁸. The endothelial derived vasoactive mediators adrenomedullin, endothelin-1, urotens....

Protocol

1. Construction of 150 mm Diameter Shear Rings (Figure 1)

NOTE: Shear rings may be constructed to create many different dimensions by varying the outer and inner Petri dish sizes, resulting in devices with different total surface areas, cell yields and developed ranges of shear forces. This report describes a 150 mm dish combined with an inner 100 mm dish for a total surface area of 98 cm² (Figure 2).

Results

Here we present representative results from both hCMEC/D3 brain endothelial cell and rat retinal microvascular endothelial cell monolayers, cultured in shear rings.

After allowing hCMEC/D3 brain endothelial cell monolayers to grow to confluence in complete EBM, the shear rings were placed on an orbital shaker for 72 hours. Using the equation from step 3.5, the calculated maximal shear stress

Discussion

The construction of the shear ring system for exposing endothelial cells to shear is a simple approach to performing shear stress studies. Nevertheless, there are a few steps that are critical for obtaining superior shear rings and better results. A complete seal should be made between the inner and outer ring to prevent media from leaking which could create inconsistent shear stress among samples. If a complete seal is not made, a minimal amount of methylene chloride should be added to the edge between the inner and out.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 x 20 mm plastic tissue culture dish	Corning	430167	The dishes must be polystyrene
150 x 25 mm plastic tissue culture dish	Corning	430599	The dishes must be polystyrene
150 mm glass Petri dish	Fisher	3160150BO
15 ml polystyrene tissue culture plastic tubes	Falcon	352099
Methylene chloride	Sigma-Aldrich	D65100
silicone rubber sealant	DAP	7079808641
ethanol	Decon	2701
3 ml transfer pipette	Becton-Dickinson	357524
printer paper
scissors
gloves
rotary tool and set	Dremel	4000-6/50
rotary tool cutting head	Dremel	EZ476
rotary tool drill head
distilled water
orbital shaker	VWR	57018-754
incubator
Rat retinal microvascular endothelial cells	Cell Biologics	RA-6065