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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Abstract

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells.

One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype.

Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

Introduction

Responses of vascular cells to their mechanical environment are important in the normal function of blood vessels and in the development of disease1. The mechanobiology of the endothelial cells (ECs) that line the interior surface of all blood vessels has been a particular focus of mechanobiological research because ECs directly experience the shear stress generated by blood flow over them. Various phenotypic changes such as inflammatory responses, altered stiffness and morphology, the release of vasoactive substances, and the localization and expression of junctional proteins depend on EC exposure to shear stress2,....

Protocol

1. Fabrication of devices and preparation of reagents

  1. Fabrication of stainless-steel module
    1. Fabricate the stainless-steel module from a grade 316 stainless-steel using a CNC milling machine according to the engineering drawing provided (Figure 1).
  2. 3D printing of a polydimethylsiloxane (PDMS) mold
    1. Prepare a 3D computer aided design (CAD) model of the PDMS mold using SolidWorks according to the engineering drawing provided (

Representative Results

Adhesion of HUVECs to regions of the well plate not coated with fibronectin was abrogated by Pluronic F-127 passivation; growth was confined to the region coated with fibronectin even after 72 h of culture, with and without shear stress application (Figure 4A, Figure 4C). Without the Pluronic F-127 passivation, HUVECs attached to the surface without fibronectin and had proliferated further by 72 h of culture (

Discussion

The swirling-well method is capable of generating complex flow profiles in a single well - Low Magnitude Multidirectional Flow (LMMF) in the center and High Magnitude Uniaxial Flow (HMUF) at the edge of the well. However, shear stress-mediated secretions of soluble mediator will be mixed in the swirling medium and affect cells in the whole well, potentially masking the true effect of a particular shear stress profile on the cells.

The coating method demonstrated here overcomes this issue by re.......

Acknowledgements

The authors gratefully acknowledge a British Heart Foundation project grant (to PDW), a National Medical Research Council Singapore TAAP and DYNAMO Grant (to XW, NMRC/OFLCG/004/2018, NMRC/OFLCG/001/2017), an A*STAR Graduate Scholarship (to KTP), and a British Heart Foundation Center of Research Excellence studentship (to MA).

....

Materials

NameCompanyCatalog NumberComments
Cell and Media
Endothelial Growth Medium (EGM-2)Lonzacc-3162
Human Umbilical Vein Endothelial CellsNANAIsolated from cords obtained from donors with uncomplicated labour at the Hammersmith Hospital
Reagents and Materials
Alexa Fuor 488-labelled goat anti-rabbit IgGThermofisher ScientificA11008
Bovine Serum AlbuminSigma-AldrichA9418-50G
Falcon 6 Well Clear Flat Bottom Not Treated Scientific Laboratory Supplies Ltd 351146
Fibronectin from Bovine PlasmaSigma-AldrichF1141-5MG
ParaformaldehydeSigma-Aldrich158127-500G
Phosphate-Buffered SalineSigma-AldrichD8537-6X500ML
Pluronic F-127Sigma-AldrichP2443
Recombinant Human TNF-aPeprotech300-01A
RS PRO 2.85 mm Black PLA 3D Printer Filament, 1 kgRS832-0264
Stainless Steel 316Metal SupermarketNA
Sylgard184 Silicone Elastomer kitFarnell101697
Triton X-100Sigma-AldrichX100-100ML
Trypsin-EDTA solutionSigma-AldrichT4049-100ML
Zonula Occludens-1 (ZO-1) antibodyCell Signaling Technology13663
DRAQ5 (5mM)Bio StatusDR50200
Equipments
Grant Orbital Shaker PSU-10iScientific Laboratory Supplies Ltd SHA7930
Leica TCS SP5 Confocal MicroscopeLeicaNA
Retaining Ring PliersMisumiRTWP32-58
Retaining Rings/Internal/C-TypeMisumiRTWS35
Ultimaker 2+3-D printerUltimakerNA
Softwares
Cura 2.6.2UltimakerNA
MATLABThe MathWorksNA
Solidworks 2016Dassault SystemesNA

References

  1. Hahn, C., Schwartz, M. A. Mechanotransduction in vascular physiology and atherogenesis. Nature Reviews Molecular Cell Biology. 10 (1), 53-62 (2009).
  2. Wang, C., Baker, B. M., Chen, C. S., Schwartz, M. A. Endothelial Cell Sensing of Flow Direction.....

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Endothelial Cells6 well PlatesOrbital ShakerMechanobiologyShear StressCell Growth SegmentationPDMS RingPluronic F 1273D PrintingCoating MethodCell CultureAtherosclerosis

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