Name: development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production
Uploaded: 2016-05-19T15:00:00
Description: Watch this Scientific Journal Video about Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production at JoVE.com

This work was supported by National Heart, Lung, and Blood Institute grants HL56237, National Institute of Diabetes and Digestive and Kidney Diseases Institute DK97391-03, National Science Foundation (NSF-1227359 and EPS-1003907).

1. Microfluidic Device Fabrication
<ol>
	<li>Standard Photolithography Fabrication of a SU-8 50 Master Mold
	<ol>
		<li>Clean the silicon wafer before spin-coating. Rinse a bare 2 inch silicon wafer with acetone for 15 min followed by isopropyl alcohol (IPA) for 15 min. Dehydrate the wafer by placing it on a hotplate at 150 &#176;C for 1 hr. After dehydration, cool the wafer at room temperature.</li>
		<li>Spin-coat the silicon wafer with SU-8 photoresist. Add 2 ml SU-8 photoresist onto the wafer. Ramp the wafer to 500...

<ol>
	<li>Curry, F. R. E., Adamson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+modulation+at+the+cell,+microvessel,+or+whole+organ+level:+towards+closing+gaps+in+our+knowledge.">Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge.</a> Cardiovasc Res. 87, 218-229 (2010).</li><li>Michel, C. C., Curry, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+permeability.">Microvascular permeability.</a> Physiol Rev. 79, 703-761 (1999).</li><li>Rogers, J. A., Nuzzo, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++entry+through+conductive+pathway+modulates+receptor-mediated+increase+in+microvessel+permeability.">Ca2+ entry through conductive pathway modulates receptor-mediated increase in microvessel permeability.</a> Am J Physiol. 271, 2377-2387 (1996).</li><li>Zhou, X., He, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+[Ca2+]i+and+caveolin-1+antagonistically+regulate+eNOS+activity+and+microvessel+permeability+in+rat+venules.">Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules.</a> Cardiovasc Res. 87, 340-347 (2010).</li><li>Zhu, L., He, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-activating+factor+increases+endothelial+[Ca2+]+i+and+NO+production+in+individually+perfused+intact+microvessels.">Platelet-activating factor increases endothelial [Ca2+] i and NO production in individually perfused intact microvessels.</a> Am J Physiol-Heart C. 288, 2869-2877 (2005).</li><li>He, P., Curry, F. 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In this article, we present detailed protocols for the development of cultured microvessel network, the characterization of EC junctions and cytoskeleton F-actin distribution, and the quantitative measurements of EC [Ca2+]i and NO production using a microfluidic device. The perfused microfluidic device provides an in vitro model that allows a close simulation of the in vivo microvascular geometries and shear flow conditions. Since the cultured microvessel network can be formed by p...

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype1,2. The microfluidics technology enables a precisely controlled fluid through a geometrically constrained microscale (sub-millimeter) channels3, which provides the opportunity for cultured cells, especially for vascular ECs, to grow under desired flow conditions. These features make the cell culture conditions closer to in vivo than the conventional, static 2D cell cultures. They are extremely important when the microfluidic devices are used to model different types of vasculatures and to study EC responses to mechanical and/or chemical stimulations.
Despite the advantages demonstrated by the microchannel network over a static cell culture, the adaptation and application of microfluidics in the biomedical field remain limited. Reported by a recent review, the majority of the publications of this field (85%) are still in engineering journals4. The performance of microfluidic devices has not been convincing enough for most biologists to switch from current techniques such as the Transwell assay and the macro-scale culture dish/glass slide to this miniaturized device. Microfluidics is a multidisciplinary field, which requires interdisciplinary collaborations to move this field forward. The objective of this technical article is to reduce the knowledge gaps between disciplines and make the fabrication procedures understandable by biologists, while providing biological application and functional validation of the microfluidic microvessels. The visualized experimental protocols include fabrication of both microfluidic devices and their biological utilities, which represents a close collaboration between engineers and biologists.
We recently reported some biological applications using the in vitro microvessel network with microfluidic device5. In order to appropriately design the dimensions of the microchannel network and apply the desired shear stress, a numerical model was built with computational fluid dynamic software to closely estimate the flow profile. Primary human umbilical vein endothelial cells (HUVECs) that were seeded into the microchannels reached confluence, i.e. covered the entire inner surfaces of the microchannel, in 3-4 days with continuous perfusion. The proper barrier formation was demonstrated by VE-cadherin staining and compared with those formed under static cell culture conditions and in intact microvessels. By applying the experimental protocols developed in individually perfused intact microvessels6-8, we quantitatively measured the changes in EC [Ca2+]i and nitric oxide (NO) production in response to adenosine triphosphate (ATP) with fluorescent indicators and confocal and conventional fluorescence microscopy. The agonist-induced increases in EC [Ca2+]i and NO production have been reported as necessary intracellular signals for inflammatory mediator-induced increases in microvessel permeability6-15. Although some previous studies showed images of DAF-2 DA loaded microfluidic devices16,17, appropriate resolution and data analysis had not yet achieved18. To our knowledge, this study demonstrates the first quantitative measurements of agonist-induced dynamic change in endothelial [Ca2+]i and NO production utilizing microfluidic based system.
Microfabrication techniques have the flexibility to fabricate microchannels down to a few microns and enable the development of complex patterns to mimic the geometries of in vivo microvasculature. Here we presented a typical microchannel network with three levels of branching. This network is fabricated by the combination of photolithography which is performed in a microfabrication cleanroom and soft lithography.

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">ATP</td>
			<td style="width:247px;">Sigma-Aldrich</td>
			<td style="width:327px;">A2383</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Fisher Scientific</td>
			<td>A929</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biopsy punch</td>
			<td>Miltex</td>
			<td>33-31 AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Albumin</td>
			<td>MP Biomedicals</td>
			<td>810014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Brain Extract (BBE)</td>
			<td>Lonza</td>
			<td>CC-4098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover-slip</td>
			<td>Fisher Scientific</td>
			<td>12-542C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAF-2 DA</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-Goat IgG (H+L) Secondary Antibody</td>
			<td>Life technologies</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DRAQ5 (nuclei staining)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>4084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endothelial Cell Growth Supplement (ECGS)</td>
			<td>Sigma-Aldrich</td>
			<td>E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluo-4 AM</td>
			<td>Life technologies</td>
			<td>F-14201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15750-078</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-548B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Pasteur pippette</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3393-10KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human umbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl alcohol (IPA)</td>
			<td>VWR</td>
			<td>89125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mammalian Ringer Solution Ingredient</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl (132 mM)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCL (4.6 mM)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2 &#183; 2H2O (2.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4 &#183;7H2O (1.2 mM)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose(5.5 mM)</td>
			<td>Fisher Scientific</td>
			<td>BP350-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaHCO3 (5.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Salt (9.1 mM)</td>
			<td>Research Organics</td>
			<td>6007H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Acid (10.9 mM)</td>
			<td>Research Organics</td>
			<td>6003H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MCDB 131 Culture Medium</td>
			<td>Life technologies</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin (F-actin staining)</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline&#160;</td>
			<td>Life technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>Exel Int</td>
			<td>29552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scotch tape</td>
			<td>3M</td>
			<td>34-8711-3070-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicon wafer</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2050 Y111072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tissue culture flasks</td>
			<td>Sigma-Aldrich</td>
			<td>Z707503-100EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Chemical Book</td>
			<td>T6878</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin Neutralizer solution</td>
			<td>Gibco</td>
			<td>R-002-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin/EDTA Solution (TE)</td>
			<td>Gibco</td>
			<td>R-001-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Cole-Parmer</td>
			<td>PTFE microbore tubing, 0.012&#34; ID x 0.030&#34; OD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VE-cadherin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-6458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Laminar hood</td>
			<td>NuAire</td>
			<td>NU-425 Class II, Type A2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD camera</td>
			<td>Hamamatsu</td>
			<td>ORCA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art</td>
			<td>F42022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>Wenesco</td>
			<td>HP-1212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Forma Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isotemp oven</td>
			<td>Barnstead</td>
			<td>3608-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithography bench</td>
			<td>Karl Suss</td>
			<td>MA6 Contact Lithography</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical microscope</td>
			<td>Nikon</td>
			<td>L200 ND &#38; Diaphod 300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shutter for the CCD camera</td>
			<td>Sutter Instrument</td>
			<td>Lambda 10-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>PVA TePla/Harrick plasma</td>
			<td>M4L/PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Brewer Science</td>
			<td>Cee 200X</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump system</td>
			<td>Harvard Apparatus</td>
			<td>703005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Software</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CAD software</td>
			<td>Autodesk</td>
			<td>AutoCAD 2015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFD simulation software</td>
			<td>COMSOL</td>
			<td>COMSOL Multiphysics 4.0.0.982</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Images acquire and analyse for NO production&#160;</td>
			<td>Universal Imaging</td>
			<td>Metafluor</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

This section shows some of the results obtained with the cultured microvessel network developed with this protocol. The microchannel pattern is a three level branching network (Figure 1A). In this design, a 159 &#181;m wide mother channel branches into two 126 &#181;m wide channels, and branches again into four 100 &#181;m wide daughter channels. A 3D numerical simulation was performed to estimate the shear stress distributions under the flow rate of 0.35 &#181;l/min (<st...

Watch this Scientific Journal Video about Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production at JoVE.com

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

The overall goal of this procedure is to develop a microfluidic device that allows endothelial cells to grow under physiologically relevant shear flow and to measure the agonist induced changes in their calcium and and nitric oxide production. This message can help advance the future of microvessel research, by validating the in vitro microvessel model and bridging the gaps between in vivo and the in vitro studies. The main advantages of this technique are the versatile design of the microchannels to mimic various vasculature and they improve cell culture environment that is closer to in vivo than the conditional static to that original cultures.Demonstrating the procedures will be Sulei and Xiang, the two from my laboratory. Before beginning, use standard photolithography to create a master mold and soft lithography to fabricate the PDMS microchannel devices as described in the text protocol. To prepare the microchannel devices, first cover the inlet and outlet with a thin piece of PDMS and place the device inside a Petri dish with a wet tissue.Then, wrap the dish with Parafilm and sterilize the device under a UV light for at least three hours. The next step is to apply the fibronectin. First, place a drop of 30 to 50 microliters of PBS at the inlet.Create a vacuum at the outlet to rinse the device. Next, place a drop of 100 micrograms per milliliter fibronectin at the inlet. Create a vacuum at the outlet to load the fibronectin solution.Next, cover the inlet and outlet. Wrap the dish with Parafilm again and incubate the device overnight at four degrees Celsius. The next day, manually rinse the device with PBS three times.Then, load it with 30 to 50 microliters of cell culture media and warm up the device in an incubator at 37 degrees Celsius for at least 15 minutes. Begin by mixing HUVECs into HUVEC culture media with eight percent Dextran at two to four million cells per milliliter. The Dextran is needed to increase the viscosity and thus improve the cell seeding.Next, put 10 to 20 microliters of cells over the inlet and introduce them using gravity or by using capillary action from a glass pasteur pipette at the outlet. Next, incubate the device for 15 to 20 minutes. Then, check the seeding status under a microscope.If necessary, load more cells to achieve a desired cell density. Once enough cells are loaded, gently rinse the device with normal, warm cell culture medium to remove the Dextran. Then, culture the device without any media perfusion for six hours.Next, set up the tubing connections for the long term perfusion. Tape the device to a Petri dish and then connect the inlet tubing to a syringe with a needle. Insert tubing into both the inlet and the outlet of the device.Connect the outlet tubing to a waste collector. Now, place the dish and waste container in an incubator. Connect the syringe with a long inlet tube to an external perfusion pump that passes through the incubator door.Then, set the perfusion rate according to the experimental design. With a well controlled perfusion, the culture in the microvessel network can be maintained up to two weeks. Therefore, it can be extended to longer term studies mimicking the cellular and the hemodynamic environment on the different physiological and the pathological condition.To begin the immuno-staining, such as VE-cadherin labeling, first fix the cells by perfusing with two percent paraformaldehyde for 30 minutes at four degrees Celsius. After a series of perfusions to block, permeabilize, and stain with antibodies, keep the device at four degrees Celsius until the cells are imaged. To image the calcium concentration in the cells, perfuse the device with 10 milligrams per milliliter of albumin in Ringer solution for 15 minutes at 37 degrees Celsius.Next, perfuse the device with five micromolar Fluo-4 AM for 40 minutes at 37 degrees Celsius. Then, wash out the lumen bound Fluo-4 AM with albumin Ringers for 15 minutes at 37 degrees Celsius. Next, start up the confocal system for intercellular calcium level imaging with Fluo-4 AM.Acquire images of the Fluo-4 loaded microvessels.Collect the baseline images for 10 minutes. Then, perfuse the device continuously with 10 micromolar ATP and record the changes of fluorescence intensity for 20 minutes. To image nitric oxide production in the cells, perfuse the device with albumin Ringers for 15 minutes at 37 degrees Celsius.Then, perfuse the cells with 5 micromolar DAF-2 DA for about 35 to 40 minutes at 37 degrees Celsius. Next, set up the fluorescence imaging system to measure ATP induced nitric oxide production. Record the baseline for 5 minutes, then perfuse the device with 10 micromolar ATP and collect the images for 30 minutes at one minute intervals.For the cell nitric oxide measurement, it is important to understand that the detach adapter for intensity profile represents a cumulative nitric oxide production with time and not a nitric oxide concentration. Manually select the region of interests from the collected images at the individual cell level. Each ROI covers the area of one individual's cell which can be indicated by the floresense outline.Quantify the ATP induced changes in endothelial calcium and nitric oxide. By calculating the changes in the floresense intensity of Fluo-4, less the tissue background. Quantify the nitrous oxide production rate by conducting the first differential conversion of the floresense intensity of DAF-2 over time.The microchannel pattern in the device has a three level branching network. A 3D numerical simulation was performed to estimate the shear stress distributions under the flow rate of 0.35 microliters per minute. Endothelial cells were seeding into the network as described.Then, F-actin and nuclei were labeled. Similarly, VE-cadherin was also stained. The results were similar to the VE-cadherin expression in an intact venule.Next, ATP induced increases in intracellular calcium and nitric oxide production were examined. Calcium levels were examined using Fluo-4 AM as described in the protocol section. Nitric oxide production was monitored using DAF-2 DA as described in the protocol section.The results were comparable to those derived with individually perfused intact venules. The advanced was easy and tightly controlled in biological conditions and the dynamic fluid environments, which would not have been possible with conventional microskill techniques. After watching this video, you should have a good understanding of how to fabricate the device, and perform calcium and nitric oxide measurements.After this technique paved the way for researchers, not only to investigate underlying agonist induced but also to open broader applications for bio medical research.

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