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 ...

<ol>
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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. 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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. 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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....

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 border="0" cellpadding="0" cellspacing="0" width="638">
	<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 par...

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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

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JoVE Journal

Bioengineering

8.8K vistas.  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. 