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).

1. Fabrication of devices and preparation of reagents
<ol>
	<li>Fabrication of stainless-steel module
	<ol>
		<li>Fabricate the stainless-steel module from a grade 316 stainless-steel using a CNC milling machine according to the engineering drawing provided (Figure 1).</li>
	</ol>
	</li>
	<li>3D printing of a polydimethylsiloxane (PDMS) mold
	<ol>
		<li>Prepare a 3D computer aided design (CAD) model of the PDMS mold using SolidWorks according to the engineering drawing provided (Figure 2).</li>
		<li>Export the CAD model to an STL file and import the STL file to Cura 2.6.2.</li>
		<li>Slice the model into layers with a print speed of 50 mm/s and infill density of 60%.</li>
		<li>Export the file as a G-code and upload it to an Ultimaker2 3D printer for printing. Use polylactic acid (PLA) as the printing material.</li>
	</ol>
	</li>
	<li>Casting of PDMS ring
	<ol>
		<li>Mix the PDMS base and curing agent (both from a silicone elastomer kit) with the ratio of 90.9% base and 9.1% curing agent.</li>
		<li>Pour approximately 2.6 mL of the well-mixed solution into the 3D printed mold.</li>
		<li>Remove bubbles in a vacuum degassing chamber.</li>
		<li>Cure it for 1 h in an 80 &#176;C furnace.</li>
		<li>Allow the PDMS ring to cool to room temperature, then remove the cured PDMS ring carefully from the mold. The engineering drawing of PDMS ring is shown in Figure 3.</li>
	</ol>
	</li>
	<li>Preparation of 1% Pluronic F-127
	<ol>
		<li>Weigh out 5 g of Pluronic F-127, pour it into a glass bottle, then add 100 mL of sterile water into the glass bottle. This gives a 5% Pluronic F-127 solution.</li>
		<li>Ensure that all Pluronic F-127 powder is submerged in the water, close the cap and autoclave it using a liquid sterilisation cycle program.</li>
		<li>After autoclave, let the solution cool to room temperature before use.</li>
		<li>Add 10 mL of 5% Pluronic F-127 solution to 40 mL of autoclaved sterile water to make 1% Pluronic F-127 solution. Perform the dilution in a biosafety cabinet (BSC) hood.</li>
		<li>Store both 1% and 5% Pluronic F-127 at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of 4% paraformaldehyde (PFA)
	<ol>
		<li>Add 800 mL of phosphate-buffered saline (PBS) to a glass beaker and heat it to 60 &#176;C whilst stirring (keep stirring from step 1.5.1 to 1.5.5).</li>
		<li>Weigh 40 g of PFA powder and add it to the warm PBS solution. 
		CAUTION: PFA is hazardous, perform steps 1.5.2 to 1.5.6 in a fume hood.</li>
		<li>Add 1 M sodium hydroxide (NaOH) slowly dropwise into the PFA solution until the solutions turns clear.</li>
		<li>Adjust the pH of the PFA solution to approximately 7.4 with 1 M of hydrochloric acid (HCl).</li>
		<li>Top up the solution to 1 L with 1X PBS. This gives 1 L of 4% PFA.</li>
		<li>Filter the PFA solution with 0.2 &#181;m filters to remove any particulates, aliquot and freeze in a -20 &#176;C freezer.</li>
	</ol>
	</li>
	<li>Preparation of 0.1% Triton-X
	<ol>
		<li>Add 50 &#181;L of pure Triton-X into 50 mL of PBS to make 0.1% Triton-X solution.</li>
	</ol>
	</li>
	<li>Preparation of 1% bovine serum albumin (BSA)
	<ol>
		<li>Weigh 0.5 g of BSA, pour it into a 50 mL centrifuge tube, and then add 50 mL of PBS into the tube.</li>
		<li>Let it roll for 1 h at room temperature on a roller to dissolve.</li>
		<li>Store 1% BSA at 4 &#176;C for up to two weeks.</li>
	</ol>
	</li>
</ol>
2. Coating of a 6-well plate
<ol>
	<li>Autoclave stainless-steel module, PDMS ring, and tweezers before use. Perform all the subsequent procedures in a BSC hood and observe aseptic techniques to ensure sterility.</li>
	<li>Place the PDMS ring in a 6-well using tweezers. Use the external rim of the PDMS ring to align the PDMS ring concentrically with the well. 
	NOTE: Only non-tissue culture treated well plates should be used.</li>
	<li>Place the stainless-steel module on top of the PDMS ring using tweezers.</li>
	<li>Insert the tips of the internal retaining ring pliers into the grip holes of the retaining ring, squeeze the holder to reduce the diameter of the retaining ring. Fit it into the 6-well, press it firmly on the stainless-steel module and release the pliers to secure the PDMS ring in the well.</li>
	<li>Add 1 mL of 5 &#181;g/mL fibronectin into the center or the edge of the well (depending on the region of interest) through the opening of PDMS ring and stainless-steel module.</li>
	<li>Swirl the plate to ensure that the fibronectin solution covers all the region of interest.</li>
	<li>Incubate for 30 min at 37 &#176;C in a humidified incubator under 95% air/5% CO2.</li>
	<li>Remove the fibronectin solution from the well and wash twice with PBS. Completely remove the PBS from the well.</li>
	<li>Remove the retaining ring, stainless-steel module, and PDMS ring from the well.</li>
	<li>Add 1.5 mL of 1% Pluronic F-127 into the center or the edge of the well (uncoated surface) and incubate for 1 h at room temperature to passivate the uncoated surface.</li>
	<li>Remove the Pluronic F-127 solution from the well and wash three times with PBS.</li>
	<li>Use the coated well immediately or store it at 4 &#176;C for up to two weeks with a layer of PBS in the coated well.</li>
</ol>
3. Seeding of HUVECs
<ol>
	<li>Use HUVECs below passage 5 in the experiment.</li>
	<li>Remove all culture medium and wash cells once with PBS.</li>
	<li>Add 3 mL of 0.05% trypsin and incubate for 3 min at 37 &#176;C in a humidified incubator under 95% air/5% CO2. Gently tap the flask to dislodge the cells. 
	NOTE: This protocol has been tested using HUVECs and the concentration of trypsin and its incubation time may be different for other type of ECs.</li>
	<li>Transfer the solution to a 15 mL centrifuge tube and neutralize the trypsin using 6 mL of culture medium (e.g., Lonza EGM-2) prewarmed to 37 &#176;C.</li>
	<li>Centrifuge at 200 x g for 5 min. Remove the neutralized trypsin solution and resuspend cells with 1 mL of prewarmed culture medium.</li>
	<li>Count the cells using a haemocytometer and seed 180k cells into a coated 6-well plate in 1.5 mL of prewarmed culture medium.</li>
	<li>Shake the well plate laterally to ensure that the cells distribute evenly in the well.</li>
	<li>Leave it in a 37 &#176;C humidified incubator under 95% air/5% CO2 overnight.</li>
	<li>Remove the unattached cells and culture medium and replace with 2mL of prewarmed culture medium. 
	&#8203;NOTE: many unattached cells floating in the medium are expected.</li>
</ol>
4. Shear stress application using an orbital shaker
<ol>
	<li>HUVECs should reach confluence after 3 days of growth. Replace the medium with 1.9 mL of prewarmed culture medium (to achieve a height of 2 mm).</li>
	<li>Place the plate on the platform of an orbital shaker in a humidified incubator under 95% air/5% CO2 and swirl it at 150 rpm for 3 days. 
	NOTE: Wipe down the exterior surface of the orbital shaker using 70% ethanol before placing it into the incubator.</li>
	<li>(Optional) After 2 days of shear, cytokines can be added to the culture medium to investigate the interplay between cytokines and shear stress. After treatment, shear the cells for another day. In this study, TNF-&#945; was used to activate the cells.</li>
	<li>Perform analyses after 3 days of shear stress application.</li>
</ol>
5. Staining and imaging of cells
<ol>
	<li>After 3 days of shear, remove the plate from incubator and wash cells twice with PBS.</li>
	<li>Fix the cells by adding 1.5 mL of 4% PFA into the well and incubate for 10 min at room temperature.</li>
	<li>Remove the 4% PFA from the well and wash twice with PBS.</li>
	<li>Permeabilize the cells by adding 0.1% Triton-X into the well and incubate for 5 min at room temperature.</li>
	<li>Remove the 0.1% Triton-X solution from the well and add 1.5 mL of 1% BSA into the well for blocking. Incubate the cells with 1% BSA for 1 h at room temperature.</li>
	<li>Dilute rabbit anti-human ZO-1 antibody at a 1:200 dilution in 1% BSA. Add 1.5 mL of diluted antibody into the well and incubate it with the cells overnight at 4 &#176;C.</li>
	<li>After overnight incubation, remove the diluted antibody and wash cells three times with PBS.</li>
	<li>Dilute Alexa Fluor 488-labelled goat anti-rabbit IgG secondary antibody at a 1:300 dilution in PBS. Add 1.5 mL of diluted secondary antibody into the well and incubate it with the cells for 1 h at room temperature.</li>
	<li>Remove the diluted secondary antibody and wash cells twice with PBS.</li>
	<li>Dilute DRAQ5 at a dilution of 1:1000 in PBS. Add 1.5 mL of diluted DRAQ5 into the well and incubate it with the cells for 15 min at room temperature to stain the cell nuclei.</li>
	<li>Remove the diluted DRAQ5 and wash three times with PBS.</li>
	<li>Perform a tile scan from the edge to the center of the well with a confocal microscope.</li>
</ol>
6. Quantification of shape index and cells number
<ol>
	<li>Post process the images using MATLAB R2016a.</li>
	<li>Read the LIF file from confocal microscope into MATLAB and convert the merged tile scan to a binary image, then threshold the image by area and intensity to distinguish nuclei from background.</li>
	<li>Subdivide the binary tile scan into 1 mm radial segments.</li>
	<li>Fit an ellipse to each individual nucleus.</li>
	<li>Count the number of ellipses within each radial segment to give a cell number.</li>
	<li>Define the shape index = as SI = 4&#960; x Area / Perimeter2. Calculate the shape index for each ellipse18.</li>
</ol>

<ol>
	<li>Hahn, C., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+in+vascular+physiology+and+atherogenesis.">Mechanotransduction in vascular physiology and atherogenesis.</a> Nature Reviews Molecular Cell Biology. 10 (1), 53-62 (2009).</li><li>Wang, C., Baker, B. M., Chen, C. S., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Cell+Sensing+of+Flow+Direction.">Endothelial Cell Sensing of Flow Direction.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (9), 2130-2136 (2013).</li><li>Tzima, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanosensory+complex+that+mediates+the+endothelial+cell+response+to+fluid+shear+stress.">A mechanosensory complex that mediates the endothelial cell response to fluid shear stress.</a> Nature. 437, 426-431 (2005).</li><li>Potter, C. M. F., Schobesberger, S., Lundberg, M. H., Weinberg, P. D., Mitchell, J. A., Gorelik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+and+compliance+of+endothelial+cells+after+shear+stress+in+vitro+or+from+different+aortic+regions:+Scanning+ion+conductance+microscopy+study.">Shape and compliance of endothelial cells after shear stress in vitro or from different aortic regions: Scanning ion conductance microscopy study.</a> PLoS ONE. 7 (2), 1-5 (2012).</li><li>Asakura, T., Karino, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Patterns+and+Spatial+Distribution+of+Atherosclerotic+Lesions+in+Human.">Flow Patterns and Spatial Distribution of Atherosclerotic Lesions in Human.</a> Circulation Research. 66 (4), 1045-1067 (1990).</li><li>Bond, A. R., Iftikhar, S., Bharath, A. A., Weinberg, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+evidence+for+a+change+in+the+pattern+of+aortic+wall+shear+stress+with+age.">Morphological evidence for a change in the pattern of aortic wall shear stress with age.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 31 (3), 543-550 (2011).</li><li>Giddens, D. P., Zarins, C. K., Glagov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fluid+mechanics+in+the+localization+and+detection+of+atherosclerosis.">The role of fluid mechanics in the localization and detection of atherosclerosis.</a> Journal of biomechanical engineering. 115, 588-594 (1993).</li><li>Schnittler, H. J., Franke, R. P., Akbay, U., Mrowietz, C., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+in+vitro+rheological+system+for+studying+the+effect+of+fluid+shear+stress+on+cultured+cells.">Improved in vitro rheological system for studying the effect of fluid shear stress on cultured cells.</a> The American journal of physiology. 265, 289-298 (1993).</li><li>Levesque, M. J., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elongation+and+orientation+of+cultured+endothelial+cells+in+response+to+shear+stress.">The elongation and orientation of cultured endothelial cells in response to shear stress.</a> Journal of biomechanical engineering. 107 (4), 341-347 (1985).</li><li>Chiu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+effect+of+disturbed+flow+on+monocytic+adhesion+to+endothelial+cells.">Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells.</a> Journal of Biomechanics. 36 (12), 1883-1895 (2003).</li><li>Venugopal Menon, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tunable+microfluidic+3D+stenosis+model+to+study+leukocyte-endothelial+interactions+in+atherosclerosis.">A tunable microfluidic 3D stenosis model to study leukocyte-endothelial interactions in atherosclerosis.</a> APL Bioengineering. 2 (1), 016103 (2018).</li><li>Warboys, C. M., Ghim, M., Weinberg, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+mechanobiology+in+cultured+endothelium:+A+review+of+the+orbital+shaker+method.">Understanding mechanobiology in cultured endothelium: A review of the orbital shaker method.</a> Atherosclerosis. 285, 170-177 (2019).</li><li>Ghim, M., Pang, K. T., Arshad, M., Wang, X., Weinberg, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+segmenting+growth+of+cells+in+sheared+endothelial+culture+reveals+the+secretion+of+an+anti-inflammatory+mediator.">A novel method for segmenting growth of cells in sheared endothelial culture reveals the secretion of an anti-inflammatory mediator.</a> Journal of Biological Engineering. 12 (1), 15 (2018).</li><li>Sage, H., Pritzl, P., Bornstein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+phenotypes+of+endothelial+cells+in+culture:+comparison+of+aortic,+venous,+capillary,+and+corneal+endothelium.">Secretory phenotypes of endothelial cells in culture: comparison of aortic, venous, capillary, and corneal endothelium.</a> Arteriosclerosis. 1 (6), 427-442 (1981).</li><li>Tunica, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+the+secretome+of+human+umbilical+vein+endothelial+cells+using+a+combination+of+free-flow+electrophoresis+and+nanoflow+LC-MS/MS.">Proteomic analysis of the secretome of human umbilical vein endothelial cells using a combination of free-flow electrophoresis and nanoflow LC-MS/MS.</a> Proteomics. 9, 4991-4996 (2009).</li><li>Griffoni, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+proteins+secreted+by+endothelial+cells+during+modeled+low+gravity+exposure.">Modification of proteins secreted by endothelial cells during modeled low gravity exposure.</a> Journal of Cellular Biochemistry. 112, 265-272 (2011).</li><li>Ghim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+three+pathways+for+macromolecule+transport+across+cultured+endothelium+and+their+modification+by+flow.">Visualization of three pathways for macromolecule transport across cultured endothelium and their modification by flow.</a> American Journal of Physiology-Heart and Circulatory Physiology. 313 (5), 959-973 (2017).</li><li>Levesque, M. J., Liepsch, D., Moravec, S., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+endothelial+cell+shape+and+wall+shear+stress+in+a+stenosed+dog+aorta.">Correlation of endothelial cell shape and wall shear stress in a stenosed dog aorta.</a> Arteriosclerosis: An Official Journal of the American Heart Association, Inc. 6 (2), 220-229 (1986).</li></ol>

The authors have nothing to disclose.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell and Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Growth Medium (EGM-2)</td>
			<td>Lonza</td>
			<td>cc-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Isolated from cords obtained from donors with uncomplicated labour at the Hammersmith Hospital</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents and Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fuor 488-labelled goat anti-rabbit IgG</td>
			<td>Thermofisher Scientific</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 6 Well Clear Flat Bottom Not Treated&#160;</td>
			<td>Scientific Laboratory Supplies Ltd&#160;</td>
			<td>351146</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from Bovine Plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F1141-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-6X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-a</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>RS PRO 2.85 mm Black PLA 3D Printer Filament, 1 kg</td>
			<td>RS</td>
			<td>832-0264</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel 316</td>
			<td>Metal Supermarket</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard184 Silicone Elastomer kit</td>
			<td>Farnell</td>
			<td>101697</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Zonula Occludens-1 (ZO-1) antibody</td>
			<td>Cell Signaling Technology</td>
			<td>13663</td>
			<td></td>
		</tr>
		<tr>
			<td>DRAQ5 (5mM)</td>
			<td>Bio Status</td>
			<td>DR50200</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Grant Orbital Shaker PSU-10i</td>
			<td>Scientific Laboratory Supplies Ltd&#160;</td>
			<td>SHA7930</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 Confocal Microscope</td>
			<td>Leica</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining Ring Pliers</td>
			<td>Misumi</td>
			<td>RTWP32-58</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining Rings/Internal/C-Type</td>
			<td>Misumi</td>
			<td>RTWS35</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimaker 2+3-D printer</td>
			<td>Ultimaker</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cura 2.6.2</td>
			<td>Ultimaker</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The MathWorks</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks 2016</td>
			<td>Dassault Systemes</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 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.

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 (<strong class="xfig"...

Watch this Scientific Journal Video about Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies at JoVE.com

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

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 ...

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

Research

JoVE Journal

Bioengineering

4.8K Views.  Imperial College London. 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.

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

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...