This work was supported by NIH/NHLBI grant R01 HL082689 to Scott I. Simon and Anthony G. Passerini.

1. Cell Culture and Substrate Preparation
 <ol>
 <li>Cut 3-inch circular substrates from a 100 x 20 mm tissue culture dish (BD Falcon) using a lathe. Sterilize substrates by submersion in 70% ethanol. Place in a Petri dish and coat with 4 ml type-I collagen (100 &mu;g/ml) for 1 hr at room temperature, then rinse with 4 ml 1 x PBS. </li>
 <li>Suspend Human Aortic Endothelial Cells (HAEC, passage 4-6) at 6.5x105 cells/ml and seed by applying 1 ml directly to substrate. Place in a 37 &deg;C, 5% CO2 incubator and allow cells to adhere to the substrate for 1 hr. </li>
 <li>Add 9 ml of Endothelial Growth Medium-2 (EGM-2) supplemented with 1x antibiotic-antimycotic solution to the cells. Change media every 2 days until the cells reach 90% confluency. </li>
 <li>Maintain THP-1 cells in RPMI 1640 medium supplemented with 10% FBS and 1x antibiotic-antimycotic solution. Passage cells when they reach a density of 106 cells/ml. </li>
</ol>
2. Cell Shearing Protocol 
Cells are conditioned in a custom cone-and-plate Cell Shearing Device (CSD). Device details and design specifications are documented in Table 1 and Figure 4.
<ol>
 <li>Prepare the CSD. Sterilize the chamber housing with ethanol and rinse with sterile PBS. Heat the housing to 37 &deg;C and level to ensure that the cone is perpendicular to the cell monolayer. Assemble the substrate with the cells from the bottom of the housing and set the cone to the desired height.</li>
 <li>Use Leibovitz-15 (L-15) medium supplemented with Endothelial BulletKit as the shearing medium to maintain proper pH in the absence of &nbsp;CO2. To stimulate inflammation, add tumor necrosis factor-&alpha; (TNF-&alpha;, 0.5 ng/ml) to the shearing medium and inject 6 ml in the device via a syringe connected to an inlet port, taking care not to introduce air bubbles in the chamber. </li>
 <li>Shear cells at a high magnitude steady shear stress (HSS, 15 dyne/cm2) or an oscillatory shear stress (OSS, 0 &plusmn; 5 dyne/cm2, 1 Hz) for 4 hr. The desired SS is achieved by rotating the cone above the cells, which is precisely controlled with a micro-stepping motor as previously described.6 The wall SS at the surface (&tau;w) is approximated by:
 
<img src="/files/ftp_upload/4169/4169eq1.jpg" alt="Equation 1" />
 
where &mu; is the viscosity of the medium, &omega; is the angular velocity of the cone, and &alpha; is the cone angle (0.5&deg;). The cone alignment with the substrate is adjusted to within 0.05&deg; through precision leveling and the gap height set at 20 &plusmn; 10 &mu;m using a depth gauge to minimize circumferential and radial variation in &tau;w. The device has been validated to ensure laminar flow characteristics. Remove cells from the device and prepare for analysis of monocyte adhesion.</li>
</ol>
3. Fabrication of PDMS Microfluidic Chamber
<ol>
 <li>Obtain master mold for desired microfluidic channel number and channel height depending on the application. The characteristics of the specific design used in this protocol are detailed in Table 1. The method for creating these is already well documented in the literature.1 Briefly, the network of channels is designed using CAD software (Autodesk Inventor) and printed at 5000 dpi on a transparency. Negative photoresist (SU8) is spun onto a silicon wafer to a thickness of 100 &mu;m. The transparency is overlaid and exposed to UV light. Non-polymerized photoresist is removed to generate the positive replica master. </li>
 <li>To make PDMS solution mix silicone base with silicone curing agent 10:1 by weight in a weigh boat and stir with a 5 ml serological pipet. Be careful not to scrape the walls of the weigh boat to avoid adding plastic flakes to the solution.</li>
 <li>Place the master wafer in a 100x15mm Petri dish and pour the PDMS solution into the dish.</li>
 <li>Cover the dish and place it into a vacuum container for 15 minutes to remove bubbles. After 15 minutes take the Petri dish out and remove any excess bubbles by gently blowing air over the surface.</li>
 <li>Place the Petri dish in an oven for 1 hour at 70 &deg;C. Remove the dish and cut out the microfluidic chambers using a sharp blade.</li>
</ol>
4. Assembly of Device on Microscope
<ol>
 <li>Turn on light source to inverted microscope and set heater to 37 &deg;C. Attach tubing to a 10 ml syringe and fill with distilled water such that there are no bubbles. Place the syringe in the syringe pump and set flow rate to induce a wall SS of 1 dyne/cm2. The wall shear stress (&tau;w) generated along the centerline of the channel is approximated using the standard equation for a parallel-plate flow chamber: 
<img src="/files/ftp_upload/4169/4169eq2.jpg" alt="Equation 2" /> 
where &mu; is the viscosity of the medium, Q is the volumetric flow rate, h is the channel height, and w is the channel width. Note: since the channels are of finite width the actual &tau;w will vary with w depending on the channel aspect ratio, and measurements are avoided at the 25% nearest the side walls.1,7</li>
 <li>Place PDMS chamber in water and use a 100&mu;l micropipette to remove air from the individual channels. Attach vacuum adapter to chamber making sure that the valve is set to off.</li>
 <li>Place Petri dish with HAEC monolayer over the objective. Place the PDMS chamber over the Petri dish and carefully set it on the HAEC monolayer while assuring that no air bubbles form between the device and the monolayer. Turn the valve so that the vacuum is on and the chamber is firmly attached.</li>
 <li>Cut 19 gauge needles blunt such that there is only 5 mm of metal left. Fill the plastic reservoir with buffer and put into PDMS chamber entrance. Repeat for appropriate number of channels to be used. Attach tubing from syringe pumps to PDMS channel exits.</li>
</ol>
5. Monocyte Adhesion Assay Under Shear Stress
<ol>
 <li>Suspend THP-1 cells at 2x106 cells/ml in HBSS + Ca2+/Mg2+ + 0.1% HSA. Activate THP-1 cells by incubating with stromal derived factor-1 (SDF-1, 50 &mu;g/ml) for 15 min at room temperature.</li>
 <li>Turn on syringe pump to establish flow, then add 50 &mu;l of cell suspension to reservoir without introducing air bubbles. Once flow is fully developed, begin data acquisition.</li>
 <li>Run for 5 min at appropriate flow rate, adding buffer to the reservoir if the solution gets low. Continue flowing buffer into the channels to wash out any unbound cells in the flow stream. </li>
</ol>
6. Data Acquisition and Analysis
<ol>
 <li>Acquire digital image sequences at 3 frames/s for 2 min following fully developed flow along the longitudinal axis of the channel at 3 locations in the channel, taking care not to record immediately after entrance or before exit ports or in the 25% nearest the side walls. </li>
 <li>Count the number of arrested cells per field by identifying their phase-bright appearance in the same focal plane as the monolayer. </li>
 <li>Firm cell arrest is defined by movement &lt; &frac12; cell diameter in 10 s. Average data over 3 random fields / channel and quantify using ImageJ software.</li>
</ol>
7. Representative Results
Although the vascular endothelium is uniformly exposed to agonists that contribute to systemic inflammation, atherosclerosis is a focal disease that develops preferentially at sites of flow disturbance in arteries, implicating spatial heterogeneity in endothelial function.8 Circulating inflammatory mediators such as TNF-&alpha; are elevated in human serum under metabolic stress and correlate with impaired endothelial function.9, 10 TNF-&alpha; induces a well-characterized response in EC that includes up-regulation of cell adhesion molecules (CAMs; e.g. VCAM-1, ICAM-1, E-selectin) and chemokines that recruit monocytes from the circulation, a hallmark of the early atherosclerotic lesion.11 Hemodynamics is an important regulator of endothelial inflammatory phenotype.12 High magnitude shear stress (HSS; e.g. 15 dynes/cm2) or pulsatile shear stress (PSS; e.g. 15 &plusmn; 5 dynes/cm2) associated with sites of resistance to atherosclerosis in arteries down-regulate TNF-&alpha;-induced responses. Conversely, low magnitude shear stress (LSS; e.g. 2 dynes/cm2) or oscillatory shear (OSS; e.g. 0 &plusmn; 5 dynes/cm2), characteristic of susceptible sites in vivo, exacerbate cytokine-induced inflammation.13, 14 Our microfluidic PDMS devices provide a platform for conducting mechanistic studies to test hypotheses related to the superposition of metabolic stress with hydrodynamic factors in the regulation of endothelial function and pathology. The utility and versatility of the approach is broadly illustrated through the following representative results. 
<img src="/files/ftp_upload/4169/4169fig1.jpg" alt="Figure 1" /> 
Figure 1. Shear stress differentially modulates THP-1 cell adhesion to TNF-&alpha; inflamed endothelial monolayers. These data were acquired using the detailed protocol described above and documented in the video. HAEC monolayers were simultaneously exposed to a low dose of the inflammatory cytokine TNF-&alpha; (0.5 ng/ml, ~EC50 for VCAM-1 up-regulation) and either HSS (15 dynes/cm2) or OSS (0 &plusmn; 5 dynes/cm2, 1 Hz) in a custom CSD for 4 hr as described in the detailed protocol. THP-1 cell arrest was quantified under shear flow in a PDMS flow chamber. This cell line that mimics the phenotype of human monocytes was chosen for its reproducibility and ease of use. HSS attenuated the inflammatory response compared to OSS, resulting in decreased THP-1 cell arrest. Data were analyzed by Student's t-test and are represented as mean &plusmn; SEM of 3 independent experiments. Adapted with permission from DeVerse et al.5
The following data demonstrate the versatility of our approach using examples from our published work. Departures from the above protocol are indicated where applicable.
<img src="/files/ftp_upload/4169/4169fig2.jpg" alt="Figure 2" /> 
Figure 2. Modulation of cytokine-induced VCAM-1 expression and monocyte recruitment in a shear stress gradient. A) HAEC monolayers were stimulated with TNF-&alpha; (0.3 ng/ml) for 4 hr under flow in a VMMC designed based on Hele-Shaw flow theory.15 A linear decrease in SS magnitude along the centerline of the channel was achieved by designing the sidewalls of the chamber to coincide with the streamlines of a two dimensional stagnation flow and shaping the end of the channel to match the iso-potential lines. The wall shear stress (&tau;w) generated along the centerline of this channel at an axial distance from the entrance (x) is given by: 
<img src="/files/ftp_upload/4169/4169eq3.jpg" alt="Equation 3" /> 

where &mu; is the viscosity of the medium, Q is the volumetric flow rate, h is the channel height, w1 is the channel entrance width, and L is the total channel length. Q was chosen to generate a gradient in SS magnitude from 16 dynes/cm2 at the inlet to 0 at the stagnation point "a" just proximal to the outlet. B)-C) VCAM-1 expression was measured in situ by immunofluorescence microscopy using antibodies conjugated to quantum dots and presented as percentage median fluorescence intensity (MFI) of unstimulated static control. SS did not modulate the basal level of VCAM-1, which is low in unstimulated HAEC (grey triangles). However, VCAM-1 expression increased relative to TNF-&alpha; at LSS magnitudes, peaking at ~2 dynes/cm2 and returning to the TNF-&alpha; baseline or below at shear magnitudes &gt; 5 dynes/cm2 (black squares). D) Monocytes (106/ml) were perfused at 2 dynes/cm2 for 5 min in parallel flow channels over monolayers preconditioned as in A). Monocyte recruitment to endothelium quantitatively correlated with the VCAM-1 expression at a given shear stress magnitude. Notably, the ability to probe VCAM-1 expression and monocyte recruitment with fine spatial resolution in a monolayer revealed significant changes over a narrow range in SS magnitude that would not be appreciated by applying only a few discrete values of shear in separate experiments. Data were analyzed by repeated measures ANOVA and differences assessed by Newman-Keuls posttest, *P &lt; 0.05 from TNF-&alpha; under static conditions. Data are mean &plusmn; SEM from 3-6 independent experiments. Adapted with permission from Tsou et al.2
<img src="/files/ftp_upload/4169/4169fig3.jpg" alt="Figure 3" /> 
 Figure 3. Triglyceride-rich lipoproteins (TGRL) modulate cytokine-induced inflammation in proportion to donor serum triglyceride levels and VCAM-1 expression. HAEC monolayers were stimulated with TNF-&alpha; (0.3 ng/ml) for 4 hr simultaneously with TGRL (10 mg/dl ApoB) isolated from human subjects after a high fat meal in static culture. A) VCAM-1 expression was measured separately in suspended EC by flow cytometry and monocyte adhesion quantified using a VMMC as described in the detailed protocol. Data are presented as % change from TNF-&alpha;. Monocyte arrest varied in direct proportion to endothelial VCAM-1 expression, which increased directly with the level of donor serum triglyceride. B) Monocyte arrest for a subset of pro-inflammatory donors was abrogated in the presence of a VCAM-1 blocking antibody. Significance was determined by paired Student's t-test, means &plusmn; SEM from 3-5 independent experiments. Adapted with permission from Wang et al.4

<ol>
	<li>Schaff, U. Y., Xing, M. M., Lin, K. K., Pan, N., Jeon, N. L., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+mimetics+based+on+microfluidics+for+imaging+the+leukocyte--endothelial+inflammatory+response.">Vascular mimetics based on microfluidics for imaging the leukocyte--endothelial inflammatory response.</a> Lab Chip. 7, 448-456 (2007).</li><li>Tsou, J. K., Gower, R. M., Ting, H. J., Schaff, U. Y., Insana, M. F., Passerini, A. G., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+inflammation+by+human+aortic+endothelial+cells+in+a+linear+gradient+of+shear+stress.">Spatial regulation of inflammation by human aortic endothelial cells in a linear gradient of shear stress.</a> Microcirculation. 15, 311-323 (2008).</li><li>Ting, H. J., Stice, J. P., Schaff, U. Y., Hui, D. Y., Rutledge, J. C., Knowlton, A. A., Passerini, A. G., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triglyceride-rich+lipoproteins+prime+aortic+endothelium+for+an+enhanced+inflammatory+response+to+tumor+necrosis+factor-alpha.">Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.</a> Circ. Res. 100, 381-390 (2007).</li><li>Wang, Y. I., Schulze, J., Raymond, N., Tomita, T., Tam, K., Simon, S. I., Passerini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+inflammation+correlates+with+subject+triglycerides+and+waist+size+after+a+high-fat+meal.">Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal.</a> Am. J. Physiol. Heart Circ. Physiol. 300, H784-H791 (2011).</li><li>Deverse, J. S., Bailey, K. A., Jackson, K. N., Passerini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+modulates+rage-mediated+inflammation+in+a+model+of+diabetes-induced+metabolic+stress.">Shear stress modulates rage-mediated inflammation in a model of diabetes-induced metabolic stress.</a> Am. J. Physiol. Heart Circ. Physiol. , (2012).</li><li>Dai, G., Kaazempur-Mofrad, M. R., Natarajan, S., Zhang, Y., Vaughn, S., Blackman, B. R., Kamm, R. D., Garcia-Cardena, G., Gimbrone, 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=Distinct+endothelial+phenotypes+evoked+by+arterial+waveforms+derived+from+atherosclerosis-susceptible+and+-resistant+regions+of+human+vasculature.">Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 14871-14876 (2004).</li><li>Young, E. W., Wheeler, A. R., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-dependent+adhesion+of+vascular+and+valvular+endothelial+cells+in+microfluidic+channels.">Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels.</a> Lab Chip. 7, 1759-1766 (2007).</li><li>Davies, P. F., Civelek, M., Fang, Y., Guerraty, M. A., Passerini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+heterogeneity+associated+with+regional+athero-susceptibility+and+adaptation+to+disturbed+blood+flow+in+vivo.">Endothelial heterogeneity associated with regional athero-susceptibility and adaptation to disturbed blood flow in vivo.</a> Semin Thromb Hemost. 36, 265-275 (2008).</li><li>Burdge, G. C., Calder, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cytokine+response+during+the+postprandial+period:+A+potential+causal+process+in+vascular+disease.">Plasma cytokine response during the postprandial period: A potential causal process in vascular disease.</a> Br. J. Nutr. 93, 3-9 (2005).</li><li>Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+metabolic+disorders.">Inflammation and metabolic disorders.</a> Nature. 444, 860-867 (2006).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+atherosclerosis.">Inflammation in atherosclerosis.</a> Nature. 420, 868-874 (2002).</li><li>Nigro, P., Abe, J., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+shear+stress+and+atherosclerosis:+A+matter+of+site+specificity.">Flow shear stress and atherosclerosis: A matter of site specificity.</a> Antioxid Redox Signal. 15, 1405-1414 (2011).</li><li>Chiu, J. J., Lee, P. L., Chen, C. N., Lee, C. I., Chang, S. F., Chen, L. J., Lien, S. C., Ko, Y. C., Usami, S., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+increases+icam-1+and+decreases+vcam-1+and+e-selectin+expressions+induced+by+tumor+necrosis+factor-[alpha]+in+endothelial+cells.">Shear stress increases icam-1 and decreases vcam-1 and e-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells.</a> Arterioscler. Thromb. Vasc. Biol. 24, 73-79 (2004).</li><li>Li, Y. S., Haga, J. H., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+effects+of+shear+stress+on+vascular+endothelial+cells.">Molecular basis of the effects of shear stress on vascular endothelial cells.</a> J. Biomech. 38, 1949-1971 (2005).</li><li>Usami, S., Chen, H. H., Zhao, Y., Chien, S., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+construction+of+a+linear+shear+stress+flow+chamber.">Design and construction of a linear shear stress flow chamber.</a> Ann. Biomed. Eng. 21, 77-83 (1993).</li><li>Helmke, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+control+of+cytoskeletal+mechanics+by+hemodynamic+forces.">Molecular control of cytoskeletal mechanics by hemodynamic forces.</a> Physiology (Bethesda). 20, 43-53 (2005).</li><li>Gower, R. M., Wu, H., Foster, G. A., Devaraj, S., Jialal, I., Ballantyne, C. M., Knowlton, A. A., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cd11c/cd18+expression+is+upregulated+on+blood+monocytes+during+hypertriglyceridemia+and+enhances+adhesion+to+vascular+cell+adhesion+molecule-1.">Cd11c/cd18 expression is upregulated on blood monocytes during hypertriglyceridemia and enhances adhesion to vascular cell adhesion molecule-1.</a> Arterioscler. Thromb. Vasc. Biol. 31, 160-166 (2011).</li><li>Wu, H., Gower, R. M., Wang, H., Perrard, X. Y., Ma, R., Bullard, D. C., Burns, A. R., Paul, A., Smith, C. W., Simon, S. I., Ballantyne, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+cd11c++monocytes+in+atherogenesis+associated+with+hypercholesterolemia.">Functional role of cd11c+ monocytes in atherogenesis associated with hypercholesterolemia.</a> Circulation. 119, 2708-2717 (2009).</li><li>Bussolari, S. R., Dewey, C. F., Gimbrone, 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=Apparatus+for+subjecting+living+cells+to+fluid+shear+stress.">Apparatus for subjecting living cells to fluid shear stress.</a> The Review of scientific instruments. 53, 1851-1854 (1982).</li></ol>

No conflicts of interest declared.

We describe the use of microfluidic PDMS devices for the on-chip assessment of endothelial inflammatory phenotype through the real-time imaging of CAM expression and monocyte adhesion. A major advantage of our approach lies in the ability to quantify outcomes associated with endothelial dysfunction in cells exposed to inflammatory mediators such as dietary lipids and cytokines under defined hydrodynamic conditions that mirror shear stress in atherogenic vessels. The VMMC facilitate the use of small amounts of reagents or...

<table border="1">
<tbody>
 <tr>
 <td>Item</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>100 x 20mm Petri Dishes</td>
 <td>BD Falcon</td>
 <td>353003</td>
 </tr>
 <tr>
 <td>Ethanol 95%</td>
 <td>EMD Chemicals</td>
 <td>EX0290-1</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Cellgro</td>
 <td>21-031-CV</td>
 </tr>
 <tr>
 <td>Type I Rat Tail Derived Collagen</td>
 <td>Gibco</td>
 <td>A10483-01</td>
 </tr>
 <tr>
 <td>Human Aortic Endothelial Cells</td>
 <td>Genlantis</td>
 <td>PH30405A</td>
 </tr>
 <tr>
 <td>Antibiotic-Antimycotic Solution</td>
 <td>Invitrogen</td>
 <td>15240-062</td>
 </tr>
 <tr>
 <td>Endothelial BulletKit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 </tr>
 <tr>
 <td>Endothelial Basal Media-2</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 </tr>
 <tr>
 <td>10 ml Syringes</td>
 <td>BD Falcon</td>
 <td>309604 </td>
 </tr>
 <tr>
 <td>Polyurethane tubing</td>
 <td>Tygon</td>
 <td>ABW0001</td>
 </tr>
 <tr>
 <td>Leibovitz-15 Media</td>
 <td>Gibco</td>
 <td>11415-069</td>
 </tr>
 <tr>
 <td>Sylgard 184 Silicone Elastomer Base</td>
 <td>Dow Corning</td>
 <td>184</td>
 </tr>
 <tr>
 <td>Sylgard 184 Silicone Elastomer Curing Agent</td>
 <td>Dow Corning</td>
 <td>184</td>
 </tr>
 <tr>
 <td>SU8 Photoresist Master Wafer</td>
 <td>UC Davis Pan Lab</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>Eclipse TE200 Inverted Microscope</td>
 <td>Nikon</td>
 <td>Eclipse TE200</td>
 </tr>
 <tr>
 <td>Syringe Pump</td>
 <td>Harvard Apparatus</td>
 <td>PHD2000</td>
 </tr>
 <tr>
 <td>19 gauge hypodermic needle</td>
 <td>Kendall</td>
 <td>8881</td>
 </tr>
 <tr>
 <td>THP-1 Monocytic Cell Line</td>
 <td>ATCC</td>
 <td>TIB-202</td>
 </tr>
 <tr>
 <td>HBSS (Hanks Buffered Saline Solution) with Ca2+/Mg2+ </td>
 <td>Gibco</td>
 <td>14025-092</td>
 </tr>
 <tr>
 <td>Tumor Necrosis Factor Alpha (TNF-&alpha;)</td>
 <td>R&amp;D Systems</td>
 <td>210-TA-010</td>
 </tr>
 <tr>
 <td>Stromal Derived Factor - 1 (SDF-1)</td>
 <td>R&amp;D Systems</td>
 <td>350-NS-010</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Cellgro </td>
 <td>10-040-CV</td>
 </tr>
 <tr>
 <td>Human Serum Albumin (HSA)</td>
 <td>ZLB Behring</td>
 <td>NDC 0053-7680-32</td>
 </tr>
 </tbody>
</table>
Table 2. Specific reagents and equipment.

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.

Watch this Scientific Journal Video about On-Chip Endothelial Inflammatory Phenotyping at JoVE.com

On-Chip Endothelial Inflammatory Phenotyping

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 ...

on-chip-endothelial-inflammatory-phenotyping

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Bioengineering

10.8K Views.  University of California, Davis. 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.

Video: On-Chip Endothelial Inflammatory Phenotyping

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

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 ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...