SynVivo technology was developed under grant #2R44HL076034 from the NHLBI.

1. Priming the SynVivo-SMN Microfluidic Device
<ol>
	<li>Each port (inlet/outlet) of the device is comprised of two parallel ports &#8211; one for flowing in surface coating moieties (adhesion molecules, growth matrices, etc.) and/or cells for seeding and the other for running the assay (Figure 1A).</li>
	<li>Completely submerge the SynVivo-SMN microfluidic device (Figure 1B) in a Petri dish containing sterile deionized (DI) water and place the dish into a vacuum desiccator. Allow the desiccator to run until all of the air is removed from the channels of the device. This should take approximately 15 min.</li>
	<li>Before removing the device from the water, place Tygon tubing (O.D. of 0.06" and I.D. of 0.02") primed with water into each port of the device with fine-point forceps. The tubing should be approximately 1 inch in length. The device can now be removed from the water. Figure 1C shows image of the device with the tubing.</li>
</ol>
2. Coating the Microfluidic Device with Desired Protein (e.g., Avidin)
<ol>
	<li>Using a pipette, place a drop of water (approximately 100 &#181;l) around the base of the one inlet port tubing. Carefully remove the tubing used to prime the device. The drop of water will prevent air from entering the device.</li>
	<li>Prepare a 1 ml syringe loaded with avidin at a concentration of 20 &#181;g/ml. Connect the syringe to a 24 G&#160;stainless steel needle and tubing. Insert the tubing to one of the inlet ports of the device. Clamp the inlet port not being utilized with a jaw clamp.</li>
	<li>Inject avidin at a flow rate of 1 &#181;l/min for 10 min to allow complete perfusion of the device. At the end of the flow time, clamp the tubing with the jaw clamp and place the device at 4 &#176;C overnight.</li>
</ol>
3. Flowing the Biotinylated Particles for Adhesion Experiments
<ol>
	<li>Allow the device to come to room temperature. Place the device on an inverted fluorescence microscope equipped with a motorized stage and a high performance camera.</li>
	<li>Prepare a solution of 2 &#181;m biotinylated particles at a concentration of 5 x 106 particles/ml in Phosphate Buffer Saline (PBS). Load the particles into a 1 ml syringe. Prepare a second 1 ml syringe of PBS only. Load each syringe on a syringe pump and connect to needle and tubing.</li>
	<li>Using a pipette, place a drop of water (approximately 100 &#181;l) around the base of the inlet port tubing. Carefully remove the tubing used to coat the device. The drop of water will prevent air from entering the device.</li>
	<li>Carefully insert the tubing's for biotinylated particles and PBS from step 3.2 into each of the inlet ports. Figure 2A shows image of the set-up.</li>
	<li>Start injecting biotinylated particles at a flow rate of 2.5 &#181;l/min. Monitor the inlet port on the microscope. At the first sign of particles, begin the timer and continue flow for 3 min.</li>
	<li>At the end of the 3 min, stop the flow of biotinylated particles while simultaneously staring the flow of PBS at a flow rate of 2.5 &#181;l/min. Allow PBS to flow in the device for 3 min to wash off unbound particles.</li>
</ol>
4. Acquiring Images and Making Area of Interest (AOI) Measurements Using Imaging Software (NIKON Elements)
<ol>
	<li>Use the "scan large image" function in the imaging software to acquire the image of the entire device.</li>
	<li>Sequentially number the bifurcations in the device and create a circular AOI with twice the diameter of the channels. In this case, set the AOI diameter to 200 &#181;m since the channel diameter is 100 &#181;m.</li>
	<li>Use the automated count function in the imaging software to export the number of particles in each AOI to an MS Excel sheet.</li>
	<li>Likewise, use the automated count feature to export the number of particles in the entire device.</li>
</ol>
5. Particle Flux Analysis Using Computational Fluid Dynamics (CFD) Models
<ol>
	<li>CFD simulations are run using commercially available software (CFD-ACE+, ESI Inc.) for the SynVivo-SMN device topology. The results are stored in a database for analyzing experimental observations. The simulation results store information on wall shear rates, velocity, particle flux, and adhesion in the device.</li>
	<li>The simulation results are used to determine the number of particles entering each AOI based on a given inlet particle concentration.</li>
</ol>
6. Generating Shear Adhesion Map
<ol>
	<li>Calculate % of adhesion by dividing the adhered particles in the bifurcation by the particles flowing in the bifurcation as shown in equation 6.1. 
	<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> where the number of particles adhered and the particles flowing are obtained from protocol steps 4.3 and 5.2, respectively.</li>
	<li>Plot the shear adhesion map using the shear rate at each bifurcation of the networks obtained from the database in step (5.1) and the % adhesion values obtained from equation 6.1.</li>
</ol>

<ol>
	<li>Weitz-Schmidt, G., Chreng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+assays.">Cell adhesion assays.</a> Methods Mol Biol. 757, 15-30 (2012).</li><li>Parsons, S. A., Jurzinsky, C., Cuvelier, S. L., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+leukocyte+recruitment+under+flow+conditions.">Studying leukocyte recruitment under flow conditions.</a> Methods Mol Biol. 946, 285-300 (2013).</li><li>Luscinskas, F. W., 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=Jr+Endothelial-dependent+mechanisms+in+chronic+inflammatory+leukocyte+recruitment.">Jr Endothelial-dependent mechanisms in chronic inflammatory leukocyte recruitment.</a> Annu Rev Med. 47, 413-421 (1996).</li><li>Adriani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preferential+targeting+of+the+diseased+microvasculature+by+disk-like+particles.">The preferential targeting of the diseased microvasculature by disk-like particles.</a> Biomaterials. 33, 5504-5513 (2012).</li><li>Decuzzi, P., 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=Flow+chamber+analysis+of+size+effects+in+the+adhesion+of+spherical+particles.">Flow chamber analysis of size effects in the adhesion of spherical particles.</a> Int J Nanomedicine. 2, 689-696 (2007).</li><li>Zou, X., 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=PSGL-1+derived+from+human+neutrophils+is+a+high-efficiency+ligand+for+endothelium-expressed+E-selectin+under+flow.">PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow.</a> Am J Physiol Cell Physiol. 289, 415-424 (2005).</li><li>Sakhalkar, H. S., 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=Leukocyte-inspired+biodegradable+particles+that+selectively+and+avidly+adhere+to+inflamed+endothelium+in+vitro+and+in+vivo.">Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo.</a> Proc Natl Acad Sci U S A. 100, 15895-15900 (2003).</li><li>Van Kruchten, R., Cosemans, J. M., Heemskerk, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+whole+blood+thrombus+formation+using+parallel-plate+flow+chambers+-+a+practical+guide.">Measurement of whole blood thrombus formation using parallel-plate flow chambers - a practical guide.</a> Platelets. 23, 229-242 (2012).</li><li>Ganguly, A., Zhang, H., Sharma, R., Parsons, S., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+umbilical+vein+endothelial+cells+and+their+use+in+the+study+of+neutrophil+transmigration+under+flow+conditions.">Isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions.</a> J Vis Exp. 66 (66), (2012).</li><li>Shirure, V. S., Reynolds, N. M., Burdick, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mac-2+binding+protein+is+a+novel+E-selectin+ligand+expressed+by+breast+cancer+cells.">Mac-2 binding protein is a novel E-selectin ligand expressed by breast cancer cells.</a> PLoS One. 7, (2012).</li><li>Ploppa, A., Schmidt, V., Hientz, A., Reutershan, J., Haeberle, H. A., Noh&#233;, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+leukocyte+distribution+during+sepsis:+an+experimental+study+on+the+interdependence+of+cell+activation,+shear+stress+and+endothelial+injury.">Mechanisms of leukocyte distribution during sepsis: an experimental study on the interdependence of cell activation, shear stress and endothelial injury.</a> Crit Care. 14, 201 (2010).</li><li>Oh, H., Diamond, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+enhances+neutrophil+membrane+tether+growth+and+slows+rolling+on+P-selectin+but+reduces+capture+from+flow+and+firm+arrest+on+IL-1-treated+endothelium.">Ethanol enhances neutrophil membrane tether growth and slows rolling on P-selectin but reduces capture from flow and firm arrest on IL-1-treated endothelium.</a> J Immunol. 181, 2472-2482 (2008).</li><li>Resto, V. A., Burdick, M. M., Dagia, N. M., McCammon, S. D., Fennewald, S. M., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-selectin-mediated+lymphocyte-cancer+cell+interactions+under+low+fluid+shear+conditions.">L-selectin-mediated lymphocyte-cancer cell interactions under low fluid shear conditions.</a> J Biol Chem. 283, 15816-15824 (2008).</li><li>Enders, S., Bernhard, G., Zakrzewicz, A., Tauber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+L-selectin+binding+by+polyacrylamide-based+conjugates+under+defined+flow+conditions.">Inhibition of L-selectin binding by polyacrylamide-based conjugates under defined flow conditions.</a> Biochim Biophys Acta. 1770, 1441-1449 (2007).</li><li>Prabhakarpandian, B., Goetz, D. J., Swerlick, R. A., Chen, X., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+functional+significance+of+adhesion+molecules+on+cultured+endothelial+cells+in+response+to+ionizing+radiation.">Expression and functional significance of adhesion molecules on cultured endothelial cells in response to ionizing radiation.</a> Microcirculation. 8, 355-364 (2001).</li><li>Brown, D. C., Larson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+to+parallel+plate+flow+chambers+to+reduce+reagent+and+cellular+requirements.">Improvements to parallel plate flow chambers to reduce reagent and cellular requirements.</a> BMC Immunology. 2, 9 (2001).</li><li>Zheng, W., Zhang, W., Jiang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+control+of+cell+adhesion+by+combination+of+surface+chemistry+and+soft+lithography.">Precise control of cell adhesion by combination of surface chemistry and soft lithography.</a> Adv Healthc Mater. 2, 95-108 (2013).</li><li>Qian, T., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro/nano-fabrication+technologies+for+cell+biology.">Micro/nano-fabrication technologies for cell biology.</a> Med Biol Eng Comput. 48, 1023-1032 (2010).</li><li>Biswas, A., Bayer, I. S., Biris, A. S., Wang, T., Dervishi, E., Faupel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+top-down+and+bottom-up+surface+nanofabrication:+techniques,+applications+&amp;+future+prospects.">Advances in top-down and bottom-up surface nanofabrication: techniques, applications &amp; future prospects.</a> Adv Colloid Interface Sci. 170, 2-27 (2012).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annu Rev Biomed Eng. 3, 335-373 (2001).</li><li>McDonald, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microfluidic+systems+in+poly(dimethylsiloxane).">Fabrication of microfluidic systems in poly(dimethylsiloxane).</a> Electrophoresis. 21, 27-40 (2000).</li><li>Prabhakarpandian, B., 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=Synthetic+microvascular+networks+for+quantitative+analysis+of+particle+adhesion.">Synthetic microvascular networks for quantitative analysis of particle adhesion.</a> Biomed Microdevices. 10, 585-595 (2008).</li><li>Rosano, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiologically+realistic+in+vitro+model+of+microvascular+networks.">A physiologically realistic in vitro model of microvascular networks.</a> Biomed Microdevices. 11, 1051-1057 (2009).</li><li>Tousi, N., Wang, B., Pant, K., Kiani, M. F., Prabhakarpandian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+adhesion+of+leukocytes+near+bifurcations+is+endothelium+independent.">Preferential adhesion of leukocytes near bifurcations is endothelium independent.</a> Microvasc Res. 80, 384-388 (2010).</li><li>Prabhakarpandian, B., 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=Bifurcations:+focal+points+of+particle+adhesion+in+microvascular+networks.">Bifurcations: focal points of particle adhesion in microvascular networks.</a> Microcirculation. 18, 380-389 (2011).</li></ol>

Publication fee&#160;for this article sponsored by&#160;CFD Research Corporation.

Parallel plate flow chambers, while providing significant insights into cell-cell and cell-particle interactions, suffer from several limitations such as high consumption of reagents and the need for multiple experimental runs to generate a shear adhesion map. The use of SynVivo-Synthetic Microvascular Networks (SynVivo-SMNs) enables the generation of a shear adhesion map from a single experiment in conditions mimicking in vivo conditions. In addition, significant savings (&#62;95%) in reagents is also obtained.
The most important step in running particle adhesion experiments with SynVivo-SMN is to ensure bubble free conditions in the chip prior to experimentation. Introduction of bubbles would lead to flow instability and lack of access to &#8220;air locked&#8221; regions of the chip. Hence, great care needs to be taken during insertions and extractions of tubings from the ports of the device. In case of a bubble found in the device during the priming step, one can repeat the priming step again to remove the bubbles. Alternatively, one can flow in media/reagents at a low flow rates (0.1 &#181;l/min or less) to ensure bubble free priming of the device.
The main limitation of the protocol is the chip being rendered unusable following the presence of a bubble that cannot be removed. However, with careful practice one can perform experiments with near 100% success. Also, the generation of shear adhesion map requires information from CFD models. However, a pre-computed database of CFD results for different flow conditions can easily overcome the need to perform the CFD simulations.
Although, the methodology presented here used the avidin-biotin system for ease of demonstration, any ligand-receptor combination on particles or cells can be used to be study particle-surface or cell-surface interactions in real-time in the device. Furthermore, cells can be cultured in the device to study particle-cell and cell-cell interactions. Culture of cells will require coating of the channels of the device with matrix suitable for desired cell types. For example, endothelial cells can be cultured on fibronectin coated channels. Following confluence, the endothelial cells can be activated using TNF-&#945; or other relevant cytokines. White Blood Cells (WBCs) can be injected and their interactions on activated endothelial cells can be studied real-time. Similar to the protocol demonstrated in this study, a shear adhesion map of white blood cells can be easily generated from a single experiment.
The protocol mentioned in this paper can be used to study effect of network morphology on particle/cell adhesion as a model of in vivo conditions. In addition, the effect of flow on particle/cell adhesion and cellular behavior for modeling physiological conditions can also be readily evaluated. Finally, the protocol can be used to study drug delivery and efficacy for targeted delivery to desired cellular populations.

Current assays to study to cell-cell and particle-cell interactions typically involve static well plate format in which particles or cells are incubated on protein matrices or adherent cells. At the end of the specified incubation time, the numbers of adherent particles or cells are quantified using microscopy1. Even though these assays provide significant insight into the biochemical processes behind these interactions, a key limitation is the lack of physiological fluid flow (typical of microcirculation) and its impact on particle adhesion.
To overcome this limitation, in vitro flow chambers have been developed in recent years. A common element of these flow chambers is a transparent apparatus perfused at low Reynolds numbers to match wall shear rates observed in blood vessels in vivo2. The vessel wall is modeled by either coating of biomolecules or growth of cells on one surface of the flow chamber3. Particles4-7 or cells8-16 are then flowed in at desired range of flow rates to quantify the number of adhering particles under various shear rates.
However, the use of parallel plate flow chambers to study and validate the biochemical phenomena is rather expensive and time consuming. This is mainly due to the fact that multiple experiments need to be conducted for generating a map of the fluidic shear vs. the number of particles/cells adhered. In addition, plate flow chambers require large volumes of reagents due to their large size (height &#62; 250 &#181;m and width &#62; 1 mm). Finally, these devices do not accurately model geometrical features (e.g., bifurcations) and flow conditions (e.g., converging vs. diverging flows) that are present in vivo.
Recent advances in lithography based microfabrication17-19 have accelerated the field of lab-on-a-chip devices20-21. These devices have been instrumental in developing a miniaturized version of the parallel plate flow chamber with dimensions in the micrometer regime. The reduction in dimension also yields significant benefits in terms of volumes of reagents, cells or particle required for experiments. However, a key limitation of the currently available devices is the use of linear channels to model microvessels, which does not mimic the complex microvasculature observed in vivo.
We have recently developed a novel methodology for recreating microvascular networks onto disposable plastic substrates resulting in synthetic representation of the in vivo conditions. These devices termed SynVivo-Synthetic Microvascular Networks (SMN) are developed using PDMS based soft-lithography process. SynVivo-SMN devices can be used to obtain shear adhesion map of cell/particle adhesion22, study targeted drug delivery23 and have been validated against in vivo data24-25. In this paper, we present a protocol that enables generation of the shear adhesion map from a single experiment in volumes as small as 1-5 &#181;l thereby resulting in significant savings of resources and time.

<table><tbody><tr><td>SynVivo-SMN</td><td>CFD Research</td><td>SMN-001</td><td>Exclusive at CFDRC</td></tr><tr><td>CFD-ACE+</td><td>ESI Inc.</td><td>N/A</td><td></td></tr><tr><td>Avidin</td><td>Invitrogen</td><td>43-4401</td><td>Any avidin source will work for this assay</td></tr><tr><td>Biotinylated Particles</td><td>Polysciences</td><td>24173-1</td><td>Any source of biotinylated particles will work for the assay</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>63018-044</td><td>Size is typical for use with SynVivo-SMN</td></tr><tr><td>NIKON Elements</td><td>NIKON Instruments</td><td>N/A</td><td>Any other imaging software can be used</td></tr></tbody></table>

generation of shear adhesion map using synvivo synthetic microvascular networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Figure 1A shows a schematic and a bright field image of SynVivo-SMN device. Figure 1B shows the SynVivo-SMN device mounted on a glass slide. Figure 1C shows the device with tubing following priming with water in a vacuum desiccator.
Figure 2A shows an image of the experimental-set up. Figure 2B shows a typical avidin-coated SynVivo-SMN device following binding of 2 &#181;m biotinylated particles. Note that particles preferentially adhere near the bifurcations in the network.
Figure 3A shows the numbered bifurcations in the SynVivo-SMN network. Figure 3B shows sample wall shear map generated by the CFD model of the device. The shear rate varies in the device ranging from 250 sec-1 to 15 sec-1 as observed in the microvasculature in vivo. Note that these varying shear rates cannot be obtained simultaneously in linear microfluidic channels as they provide a constant shear rate for each flow rate. Figure 3C shows a plot of the values of the shear rate at each of the bifurcations of the network. The data shows that the shear rate patterns are complex and cannot be obtained with a simple analytical relationship unlike linear flow channels. Furthermore, multiple bifurcations (AOIs) are present in the network that fall in the same shear bin, adding to the statistical confidence of the data.
Figure 4A shows sample results of the CFD analysis for particle fluxes in the network which are used to compute the particle fluxes in the branches and bifurcations of the experimental network. Figure 4B shows the shear adhesion map computed from the single SynVivo-SMN experiment. The shear adhesion maps follows the inverse relationship as observed from shear adhesion map obtained from linear channel experiments. However, in contrast using a SynVivo assay, a single experiment allows generation of this shear map unlike multiple experimental runs required from the linear channels resulting in significant savings of time and resources. Note that experiments are run in triplicates for maximal statistical analysis.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51025/51025fig1highres.jpg" width="500" /> 
Figure 1. SynVivo-SMN devices. Left panel (Figure 1A) shows a schematic of the device. Right panel (Figure 1A) shows bright field image of the device. Figure 1B shows the SynVivo-SMN device mounted on microscope glass slide. Figure 1C shows the device with Tygon&#8482; tubing attached to the inlet/outlet ports following priming with water.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51025/51025fig2highres.jpg" width="500" /> 
Figure 2. Typical adhesion assay in SynVivo-SMN. Figure 2A shows an image of the experimental-set up. Figure 2B shows biotinylated particles following binding in the device. Note that particle adhesion is found to be localized near bifurcation compared to the branches of the network.
<img alt="Figure 3" fo:content-width="4.5in" src="/files/ftp_upload/51025/51025fig3highres.jpg" width="500" /> 
Figure 3. Shear analysis in SynVivo-SMN. Figure 3A shows all the bifurcations numbered in the network and a magnified view of bifurcations from the experiments. Figure 3B shows the qualitative wall shear map in the bifurcations in the network. Figure 3C shows the quantitative information on shear in the branches in the network. Note that the shear patterns in the network are complex and span the physiological ranges of shear rates (0-240 sec-1) found in vivo.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51025/51025fig4highres.jpg" width="500" /> 
Figure 4. Generation of shear adhesion map. Figure 4A highlights the particle trajectories (shown in white) in the networks obtained from the CFD simulation. These trajectories are post-processed to obtain particle fluxes in the different branches and the bifurcations. Figure 4B shows the shear adhesion map obtained from a single SynVivo-SMN experiment.

Watch this Scientific Journal Video about Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks at JoVE.com

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

generation-shear-adhesion-map-using-synvivo-synthetic-microvascular

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K Views.  CFD Research Corporation. Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables. 

Video: Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

On-Chip Endothelial Inflammatory Phenotyping

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

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

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

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Developmental Biology

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Cancer Research

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo

The importance of sharing experimental data in neuroscience grows with the amount and complexity of data acquired and various techniques used to obtain and process these data. However, the majority of experimental data, especially from individual studies of regular-sized laboratories never reach wider research community. A graphical user interface (GUI) engine called Neurovascular Network Explorer 2.0 (NNE 2.0) has been created as a tool for simple and low-cost sharing and exploring of vascular imaging data. NNE 2.0 interacts with a database containing optogenetically-evoked dilation/constriction time-courses of individual vessels measured in mice somatosensory cortex in vivo by 2-photon microscopy. NNE 2.0 enables selection and display of the time-courses based on different criteria (subject, branching order, cortical depth, vessel diameter, arteriolar tree) as well as simple mathematical manipulation (e.g. averaging, peak-normalization) and data export. It supports visualization of the vascular network in 3D and enables localization of the individual functional vessel diameter measurements within vascular trees.
NNE 2.0, its source code, and the corresponding database are freely downloadable from UCSD Neurovascular Imaging Laboratory website1. The source code can be utilized by the users to explore the associated database or as a template for databasing and sharing their own experimental results provided the appropriate format.

A graphical user interface for exploring and sharing a database of optogenetically-induced vascular responses in mouse somatosensory cortex in vivo measured by 2-photon microscopy is presented. It allows browsing the data, criteria-based selection, averaging, localization of measurements within a 3D volume of vasculature and exporting the data.

The importance of sharing experimental data in neuroscience grows with the amount and complexity of data acquired and various techniques used to obtain ...

Neuroscience

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 ...