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 (<strong cl...

<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 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 ...

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for sheer stress application using the orbital shaker model. A common way to study effects of flow on endothelium is to culture endothelial cells in multi-well culture plates on an orbital shaker. The orbital shaker induces a wave that rotates around the well.Cells at the center of the well experience the kind of flows that are thought to lead to atherosclerosis. Cells closer to the edge experience flow that is thought to be protective. Properties of the cells in each region are assumed to depend on the sheer stresses that they experience.However, there is a potential floor in this logic. Endothelial cells release mediators in a flow-dependent fashion. These mediators will reach a uniform concentration in the swirling medium and will therefore affect cells in regions other than the one where they were released.That may corrupt or hide true effects of shear on cell behavior. The effect is not limited to the swirling well system. It may occur even in simple models, like the parallel plate flow chamber.It can be avoided by growing cells in only one part of the system. Here, we describe methods for permitting cell adhesion only in the center or only at the edge of the swirling well. Fabrication of stainless steel module.Fabricate the stainless steel module from a grade 316 stainless steel according to an engineering drawing provided. 3D printing on PDMs. Prepare a 3D CAD model of PDMs mold using SOLIDWORKS according to the engineering drawing provided.Export the CAD model to an STL file and import the STL file into Cura 2.6.2. Slice the model into layers with a print speed of 50 millimeters per second and infill density of 60%Export the file as a GCode. Uploaded it to an Ultimaker 3D printer for printing.Use polylactic acid as the printing material. Casting of PDMS ring. Mix the PDMS base and curing agent well with the ratio of 90.9%base and 9.1%curing agent.Pour the well-mixed solution into the 3D-printed mold. Remove bubbles in a vacuum degassing chamber. Cure it for one hour in an 80-degrees furnace.Allow the PDMS ring to cool to room temperature. Then remove the cured PDMS ring carefully from the mold. Preparation of 1%Pluronic F-127.Weigh out five grams of Pluronic F-127. Pour it into a glass bottle. Then add a hundred mil of Milli-Q water into the glass bottle.This gives a 5%Pluronic F-127 solution. Ensure that all Pluronic F-127 powder is submerged in the water. Close the cap and autoclave it using a liquid sterilization cycle program.Let the solution cool to room temperature before use. Add 10 mil of 5%Pluronic F-127 solution to 40 mil of autoclaved Milli-Q water to make 1%Pluronic F-127 solution. Perform the dilution in a biosafety cabinet hood.Store both 1%and 5%Pluronic F-127 at room temperature. Coating of 6-well plate. Autoclave stainless steel module, PDMS ring, and tweezers before use.Perform all subsequent procedures in a BSC hood and observe aseptic techniques to ensure sterility. Place the PDMS ring in a 6-well using tweezers. Use the external rim of the PDMS ring to align the PDMS ring concentrically within the well.Note, only non tissue culture-treated well plates should be used. Place the stainless steel module on top of the PDMS ring using tweezers. Insert the tips of the internal retaining ring pliers into the grip holds of the retaining ring.Squeeze the holder to reduce the diameter of the retaining ring. Put 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. Add one mil of five microgram per microliter 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.Swirl the plate to ensure the fibronectin solution covers all the region of interest. Incubate for 30 minutes at 37 degrees in a humidified incubator under 95%air and 5%CO2. Remove the fibronectin solution from the well.And now wash twice with PBS. Once done, completely remove the PBS from the well. Remove the retaining ring, stainless steel module, and PDMS ring from the well.Add 1.5 mil of 1%Pluronic F-127 into the well and incubate for one hour at room temperature to passivate the uncoated surface. Remove the Pluronic F-127 solution from the well and wash three times with PBS. Once washed, use the coated well immediately or store at four degrees for up to two weeks with the layer of PBS in the coated well.Seeding of endothelial cells. Count cells using a hemocytometer and seed 180, 000 cells into a coated 6-well plate in 1.5 mil of pre-warm cell culture medium. Shake the well plate laterally to ensure that the cells distribute evenly in the well.Leave it in a 37-degrees humidified incubator under 95%air and 5%CO2 overnight. Shear stress application using an orbital shaker. Cells should reach confluence after three days of growth.Replace the medium with 1.9 mil of pre-warn cell culture medium to achieve a height of two millimeters. Place a well plate on the platform of an orbital shaker in a humidified incubator and swirl it at 150 RPM for three days. Optional, after two days of shear, cytokines can be added to the cell culture medium to investigate the interplay between cytokines and sheer stress.After treatment, shear the cells for another day. In this study, TNF-alpha was used to activate the cells. Perform analysis after three days of shear stress application.Microscope images show that Pluronic F-127 prevented HUVEC adhesion to the region without fibronectin coating. No HUVECs were attached to the part of the well surface that had not been pretreated with fibronectin prior to passivation of Pluronic F-127 after 24 hours, in figure A, and 72 hours, in figure C, of growth. Without Pluronic F-127 passivation, HUVECs were attached to the surface without fibronectin, 24 hours after seeding, in figure B, and are proliferated further by 72 hours, in figure D.The scale bar is equal to 500 micrometers.Nuclear Red stain shows the morphology of sheared HUVECs, A, in the center, and B, at the edge of a full well. Scale bar is equal to a hundred micrometers. A and B also show cell outlines delineated by immunostaining of ZO-1.Note the alignment and elongation of cells at the edge but not at the center. It shows no significant difference in nuclear shape index, indicating roundness, between HUVECs grown in full well and segmented wells were seen for untreated or TNF-alpha-treated HUVECs. Cells were more elongated near the edge of the well.A tendency for a greater elongation in TNF-alpha-treated HUVECs was not consistently significant across locations. No significant difference was observed between full and segmented wells in density number of A, untreated, and B, TNF-alpha-treated HUVECs at different radial locations. In both cases, there were more cells per unit area at the edge than at the center of the well.In conclusion, the method we have outlined here allows you to grow cells in just one region of the swirling well or indeed, of any plastic-based cell culture system. That means cells in one part of the well experiencing one type of flow are not influenced by mediators released from cells in another region experiencing different flows. Not only that, we can look at cells grown in one region with or without cells being grown in other regions.That allows the effects of such soluble mediators to be demonstrated. Testing the effects of conditioned medium on naive cells permits the same thing. By analyzing condition medium, the mediators can be identified.

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

Research

JoVE Journal

Bioengineering

4.7K visualizações.  Imperial College London. Este protocolo descreve um método de revestimento para restringir o crescimento de células endoteliais a uma região específica de uma placa de 6 poços para aplicação de estresse pura usando o modelo de agitador orbital. Uma maneira comum de estudar os efeitos do fluxo no endotélio é cultivar células endoteliais em placas de cultura multi-bem em um agitador orbital. O agitador orbital induz uma onda que gira ao redor do poço.As células no centro do poço experimentam o tipo ...