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Method Article
* These authors contributed equally
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.
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.
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 stress2,3,4. Shear-dependent endothelial properties may also account for the patchy development of diseases such as atherosclerosis5,6,7.
It is useful to study the effect of shear on ECs in culture, where stresses can be controlled, and ECs can be isolated from other cell types. Commonly used in vitro devices for applying shear stress to ECs include the parallel-plate flow chamber and the cone-and-plate viscometer, but only uniaxial steady, oscillatory, and pulsatile flow can be applied8,9. Although modified flow chambers with tapered or branching geometries and microfluidic chips that mimic a stenotic geometry have been developed, their low-throughput and the relatively short culture duration that is possible pose a challenge10, 11.
The orbital shaker (or swirling well) method for the study of endothelial mechanotransduction, in which cells are grown in standard cell culture plasticware placed on the platform of an orbital shaker, is gaining increasing attention because it is capable of chronically imposing complex, spatially varying shear stress patterns on ECs with high throughput (see review by Warboys et al.12). Computational Fluid Dynamics (CFD) simulations have been employed to characterize the spatial and temporal variation of shear stress in a swirling well. The swirling motion of culture medium caused by the orbital motion of the shaker platform on which the plate is placed leads to Low Magnitude Multidirectional Flow (LMMF, or putatively pro-atherogenic flow) at the center and High Magnitude Uniaxial Flow (HMUF, or putatively atheroprotective flow) at the edge of the wells of a 6-well plate. For example, time-averaged wall shear stress (TAWSS) is approximately 0.3 Pa at the center and 0.7 Pa at the edge of a 6-well plate swirled at 150 rpm with a 5 mm orbital radius13. The method requires only commercially available plasticware and the orbital shaker itself.
There is, however, a drawback to the method (and to other methods of imposing flows in vitro): ECs release soluble mediators and microparticles in a shear-dependent manner14,15,16 and this secretome may affect ECs in regions of the well other than the one in which they were released, due to the mixing in the swirling medium. This may mask the actual effects of shear stress on EC phenotype. For example, Ghim et al. have speculated that this accounts for the apparently identical influence of different shear profiles on transcellular transport of large particles17.
Here we describe a method for promoting human umbilical vein endothelial cell (HUVEC) adhesion in specific regions of a 6-well plate using fibronectin coating while using Pluronic F-127 to passivate the surface and prevent growth elsewhere. The method resolves the limitation described above because, by segmenting cell growth, ECs experience only one kind of shear profile, and are not influenced by secretomes from ECs exposed to other profiles elsewhere in the well.
1. Fabrication of devices and preparation of reagents
2. Coating of a 6-well plate
3. Seeding of HUVECs
4. Shear stress application using an orbital shaker
5. Staining and imaging of cells
6. Quantification of shape index and cells number
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 (
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...
The authors have nothing to disclose.
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).
Name | Company | Catalog Number | Comments |
Cell and Media | |||
Endothelial Growth Medium (EGM-2) | Lonza | cc-3162 | |
Human Umbilical Vein Endothelial Cells | NA | NA | Isolated from cords obtained from donors with uncomplicated labour at the Hammersmith Hospital |
Reagents and Materials | |||
Alexa Fuor 488-labelled goat anti-rabbit IgG | Thermofisher Scientific | A11008 | |
Bovine Serum Albumin | Sigma-Aldrich | A9418-50G | |
Falcon 6 Well Clear Flat Bottom Not Treated | Scientific Laboratory Supplies Ltd | 351146 | |
Fibronectin from Bovine Plasma | Sigma-Aldrich | F1141-5MG | |
Paraformaldehyde | Sigma-Aldrich | 158127-500G | |
Phosphate-Buffered Saline | Sigma-Aldrich | D8537-6X500ML | |
Pluronic F-127 | Sigma-Aldrich | P2443 | |
Recombinant Human TNF-a | Peprotech | 300-01A | |
RS PRO 2.85 mm Black PLA 3D Printer Filament, 1 kg | RS | 832-0264 | |
Stainless Steel 316 | Metal Supermarket | NA | |
Sylgard184 Silicone Elastomer kit | Farnell | 101697 | |
Triton X-100 | Sigma-Aldrich | X100-100ML | |
Trypsin-EDTA solution | Sigma-Aldrich | T4049-100ML | |
Zonula Occludens-1 (ZO-1) antibody | Cell Signaling Technology | 13663 | |
DRAQ5 (5mM) | Bio Status | DR50200 | |
Equipments | |||
Grant Orbital Shaker PSU-10i | Scientific Laboratory Supplies Ltd | SHA7930 | |
Leica TCS SP5 Confocal Microscope | Leica | NA | |
Retaining Ring Pliers | Misumi | RTWP32-58 | |
Retaining Rings/Internal/C-Type | Misumi | RTWS35 | |
Ultimaker 2+3-D printer | Ultimaker | NA | |
Softwares | |||
Cura 2.6.2 | Ultimaker | NA | |
MATLAB | The MathWorks | NA | |
Solidworks 2016 | Dassault Systemes | NA |
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