We thank Dr. Pina Colarusso and the Live Cell Imaging Facility for their assistance with imaging and image analysis; Ms. Lailey for her technical assistance; and unit 51 at the Foothills Hospital in Calgary, AB for providing human umbilical cords. Dr. K.D. Patel is an Alberta Innovates: Health Solutions Scientist. This work was supported by an operating grant from the Canadian Institutes for Health Research and equipment and infrastructure grants from the Canadian Foundation for Innovation and the Alberta Science and Research Authority.

1. Isolation and Maintenance of Human Vascular Endothelial Cells
<ol>
 <li>Level 2 biosafety procedures must be used when working with human blood and tissue. Using scissors cut the cord from the placenta and then closely examine the cord for blood clots and damage caused by clamping the cord during delivery. Cut out and discard these portions of the cord.</li>
 <li>Prepare fresh collagenase solution at 1 mg/ml by pouring 50 ml of warm cord buffer into a tube that contains 50 mg collagenase.</li>
 <li>Change into steri...

<ol>
	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nat. Rev. Immunol. 6, 173-182 (2006).</li><li>Diacovo, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+rolling,+arrest,+and+transmigration+across+activated,+surface-adherent+platelets+via+sequential+action+of+P-selectin+and+the+beta+2-integrin+CD11b/CD18.">Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18.</a> Blood. 88, 146-157 (1996).</li><li>Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat. Rev. Immunol. 7, 678-689 (2007).</li><li>Petri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+LSP1+is+involved+in+endothelial+dome+formation,+minimizing+vascular+permeability+changes+during+neutrophil+transmigration+in+vivo.">Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo.</a> Blood. 117, 942-952 (2011).</li><li>Chavakis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+junctional+adhesion+molecule-C+promotes+neutrophil+transendothelial+migration+in+vitro+and+in+vivo.">The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo.</a> J. Biol. Chem. 279, 55602-55608 (2004).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Isolation+Protocol.">Neutrophil Isolation Protocol.</a> J. Vis. Exp. (17), e745 (2008).</li><li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+leukocyte+transmigration:+cell+surface+interactions+and+signaling+events.">Regulation of leukocyte transmigration: cell surface interactions and signaling events.</a> J. Immunol. 172, 7-13 (2004).</li><li>Alcaide, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+under+shear+flow:+it's+all+about+endothelial+cell+rings+and+gaps.">Neutrophil recruitment under shear flow: it's all about endothelial cell rings and gaps.</a> Microcirculation. 16, 43-57 (2009).</li><li>Jutila, 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=Measurement+of+neutrophil+adhesion+under+conditions+mimicking+blood+flow.">Measurement of neutrophil adhesion under conditions mimicking blood flow.</a> Neutrophil Methods and Protocols. 412, 239-256 (2007).</li><li>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+rolling+and+adhesion+in+vitro+under+flow+conditions.">Studying leukocyte rolling and adhesion in vitro under flow conditions.</a> Basic Cell Culture Protocols. 290, 331-342 (2005).</li><li>Wiese, G., Barthel, S. R., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Physiologic+E-Selectin-Mediated+Leukocyte+Rolling+on+Microvascular+Endothelium.">Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium.</a> J. Vis. Exp. (24), e1009 (2009).</li><li>Petzelbauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J. Immunol. 151, 5062-5072 (1993).</li><li>Bonder, C. S., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+leukocyte+recruitment+to+splanchnic+organs+to+reduce+inflammation.">Modulating leukocyte recruitment to splanchnic organs to reduce inflammation.</a> Am J Phys - Gastrointestinal and Liver Phys. 284, G729-G733 (2003).</li><li>Cuvelier, 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=Eosinophil+adhesion+under+flow+conditions+activates+mechanosensitive+signaling+pathways+in+human+endothelial+cells.">Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells.</a> J. Exp. Med. 202, 865-876 (2005).</li><li>Massia, S. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cell+interactions+with+surface-coupled+adhesion+peptides+on+a+nonadhesive+glass+substrate+and+two+polymeric+biomaterials.">Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.</a> J. Biomed. Mat. Res. 25, 223-242 (1991).</li></ol>

No conflicts of interest declared.

Researchers have routinely used endothelial cells from different vascular beds to study neutrophil recruitment and transmigration. Examples include, but are not limited to, dermal microvascular endothelial cells12, liver sinusoidal endothelial cells13 and endothelial cells from human umbilical vein10. Among them HUVEC gained wide popularity because of their relative ease of isolation and availability. HUVEC are a powerful in vitro model system that mimics many endothelial process...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent and Equipment</td>
			<td>Company</td>
			<td>Catalogue number </td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Worthington</td>
			<td>4197</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cord buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Composition: 140 mM NaCl 4 mM KCl 10 mM D-glucose in 1mM NaH2PO4/Na2HPO4 buffer at pH 7.4</td>
		</tr>
		<tr>
			<td>Endothelial Cell Media (ECM)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>M199 with eagle salts supplemented with 16% human serum containing 100 units of penicillin 100 &mu;g of streptomycin and 0.3 mg of L-glutamine/ml</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>GIBCO</td>
			<td>31100-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Glutamate (100X)</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ibidi chambers</td>
			<td>Ibidi</td>
			<td>80606</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TNF-&alpha;</td>
			<td>Prepro Tech</td>
			<td>300-01A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human Albumin 20% solution</td>
			<td>Gemini Bioproducts</td>
			<td>800120050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS without Ca2+ and Mg2+</td>
			<td>Sigma</td>
			<td>H2487-10X</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS with Ca2+ and Mg2+</td>
			<td>Sigma</td>
			<td>H1387-10X</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

Watch this Scientific Journal Video about Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions at JoVE.com

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

isolation-human-umbilical-vein-endothelial-cells-their-use-study

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Immunology and Infection

17.0K 조회수.  University of Calgary. 이 문서는 먼저 제대 정맥 내피 세포로부터 인간을 분리하기위한 절차를 설명하고 유동 조건 하에서 호중구 윤회를 검사하기 위해 이러한 세포를 사용하는 방법을 보여줍니다. 유리의 광학 특성을 가진 고분자로 만들어진 낮은 볼륨 흐름 챔버를 사용하여 희귀 세포 집단의 살아있는 세포 형광 이미징도 가능합니다.