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>

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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. 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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><tbody><tr><td>&lt;strong&gt;Cell and Media&lt;/strong&gt;</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>&lt;strong&gt;Reagents and Materials&lt;/strong&gt;</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&amp;nbsp;</td><td>Scientific Laboratory Supplies Ltd&amp;nbsp;</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>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Grant Orbital Shaker PSU-10i</td><td>Scientific Laboratory Supplies Ltd&amp;nbsp;</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>&lt;strong&gt;Softwares&lt;/strong&gt;</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"...

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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

4.9K Views.  Imperial College London. Dit protocol beschrijft een coatingmethode om endotheelcelgroei te beperken tot een specifiek gebied van een 6-put plaat voor pure stresstoepassing met behulp van het orbitale shakermodel. Een veel voorkomende manier om effecten van stroming op endotheel te bestuderen, is door endotheelcellen in multi-well kweekplaten op een orbitale shaker te gekweekt. De orbitale shaker induceert een golf die rond de put draait.Cellen in het midden van de put ervaren het soort stromen waarvan wordt g...

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

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Biology

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Cancer Research

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Developmental Biology

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 Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Medicine

3D Bioprinting for Rapid Vascular Network Fabrication Using Vessels-on-a-Plate

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Author Spotlight: Automated Bioprinting for High-Throughput Vascular Model Fabrication

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

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

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

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

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

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

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