Name: a cell culture model of resistance arteries
Uploaded: 2017-09-08T12:00:00
Description: Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

This work was supported by National Heart, Lung and Blood Institute grants R01088554, T32HL007284 and American Heart Association predoctoral fellowship 14PRE20420024. We especially thank the St. Jude Cellular Imaging Shared Research Core, including Randall Wakefield and Linda Horner for the electron microscopy images, Adam Straub for assistance with representative immunofluorescence images,&#160;and Pooneh Bagher for her valuable feedback on the manuscript protocol.

1. Plating Cells for the VCCC
<ol>
	<li>Culture large 225 cm2 flasks of EC and SMC under sterile conditions at 37 &#176;C, until 70-90% confluent. Ensure that more EC than SMC are used; if using primary human EC and SMC, typically 3 large flasks of EC to 1 large flask of SMC.
	<ol>
		<li>Use primary cells at lower passages and do not use past passage 10. Choose the culture media based upon cells to be co-cultured. For primary human coronary artery EC, use EBM-2 EC basal media with EGM2 MV gr...

<ol>
	<li>Isakson, B. E., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterocellular+contact+at+the+myoendothelial+junction+influences+gap+junction+organization.">Heterocellular contact at the myoendothelial junction influences gap junction organization.</a> Circ Res. 97 (1), 44-51 (2005).</li><li>Little, T. L., Xia, J., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+tracers+define+differential+endothelial+and+smooth+muscle+coupling+patterns+within+the+arteriolar+wall.">Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall.</a> Circ Res. 76 (3), 498-504 (1995).</li><li>Heberlein, K. R., Straub, A. C., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myoendothelial+junction:+breaking+through+the+matrix?.">The myoendothelial junction: breaking through the matrix?.</a> Microcirculation. 16 (4), 307-322 (2009).</li><li>Straub, A. C., Zeigler, A. C., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myoendothelial+junction:+connections+that+deliver+the+message.">The myoendothelial junction: connections that deliver the message.</a> Physiology (Bethesda). 29 (4), 242-249 (2014).</li><li>Isakson, B. E., Ramos, S. I., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++and+inositol+1,4,5-trisphosphate-mediated+signaling+across+the+myoendothelial+junction.">Ca2+ and inositol 1,4,5-trisphosphate-mediated signaling across the myoendothelial junction.</a> Circ Res. 100 (2), 246-254 (2007).</li><li>Dora, K. A., Doyle, M. P., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+intracellular+calcium+in+smooth+muscle+causes+endothelial+cell+generation+of+NO+in+arterioles.">Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.</a> Proc Natl Acad Sci U S A. 94 (12), 6529-6534 (1997).</li><li>Sonkusare, S. K., 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=Elementary+Ca2++signals+through+endothelial+TRPV4+channels+regulate+vascular+function.">Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function.</a> Science. 336 (6081), 597-601 (2012).</li><li>Ledoux, J., 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=Functional+architecture+of+inositol+1,4,5-trisphosphate+signaling+in+restricted+spaces+of+myoendothelial+projections.">Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections.</a> Proc Natl Acad Sci U S A. 105 (28), 9627-9632 (2008).</li><li>Boerman, E. M., Everhart, J. E., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+age+decreases+local+calcium+signaling+in+endothelium+of+mouse+mesenteric+arteries+in+vivo.">Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo.</a> Am J Physiol Heart Circ Physiol. 310 (9), H1091-H1096 (2016).</li><li>Toussaint, F., Charbel, C., Blanchette, A., Ledoux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+regulates+intracellular+Ca(2)(+)+dynamics+in+native+endothelial+cells.">CaMKII regulates intracellular Ca(2)(+) dynamics in native endothelial cells.</a> Cell Calcium. 58 (3), 275-285 (2015).</li><li>Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Cell+Vascular+Smooth+Muscle+Cell+Co-Culture+Assay+For+High+Throughput+Screening+Assays+For+Discovery+of+Anti-Angiogenesis+Agents+and+Other+Therapeutic+Molecules.">Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules.</a> Int J High Throughput Screen. 2010 (1), 171-181 (2010).</li><li>Biwer, L. A., 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=Two+functionally+distinct+pools+of+eNOS+in+endothelium+are+facilitated+by+myoendothelial+junction+lipid+composition.">Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition.</a> Biochim Biophys Acta. 1861 (7), 671-679 (2016).</li><li>Straub, A. 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=Endothelial+cell+expression+of+haemoglobin+alpha+regulates+nitric+oxide+signalling.">Endothelial cell expression of haemoglobin alpha regulates nitric oxide signalling.</a> Nature. 491 (7424), 473-477 (2012).</li><li>Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+expression+of+an+Ins(1,4,5)P3+receptor+at+the+myoendothelial+junction+selectively+regulates+heterocellular+Ca2++communication.">Localized expression of an Ins(1,4,5)P3 receptor at the myoendothelial junction selectively regulates heterocellular Ca2+ communication.</a> J Cell Sci. 121 (Pt 21), 3664-3673 (2008).</li><li>Axel, D. I., 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=Induction+of+cell-rich+and+lipid-rich+plaques+in+a+transfilter+coculture+system+with+human+vascular+cells.">Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells.</a> J Vasc Res. 33 (4), 327-339 (1996).</li><li>Hastings, N. E., Simmers, M. B., McDonald, O. G., Wamhoff, B. R., Blackman, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis-prone+hemodynamics+differentially+regulates+endothelial+and+smooth+muscle+cell+phenotypes+and+promotes+pro-inflammatory+priming.">Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming.</a> Am J Physiol Cell Physiol. 293 (6), C1824-C1833 (2007).</li><li>Herzog, D. P., Dohle, E., Bischoff, I., Kirkpatrick, 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=Cell+communication+in+a+coculture+system+consisting+of+outgrowth+endothelial+cells+and+primary+osteoblasts.">Cell communication in a coculture system consisting of outgrowth endothelial cells and primary osteoblasts.</a> Biomed Res Int. , 320123 (2014).</li><li>Jacot, J. G., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+injury+induces+vascular+smooth+muscle+cell+proliferation+in+highly+localized+regions+of+a+direct+contact+co-culture+system.">Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.</a> Cell Biochem Biophys. 52 (1), 37-46 (2008).</li><li>Scott-Drechsel, D., 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+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li><li>van Buul-Wortelboer, M. F., 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=Reconstitution+of+the+vascular+wall+in+vitro.+A+novel+model+to+study+interactions+between+endothelial+and+smooth+muscle+cells.">Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.</a> Exp Cell Res. 162 (1), 151-158 (1986).</li><li>Wilhelm, I., Fazakas, C., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier.">In vitro models of the blood-brain barrier.</a> Acta Neurobiol Exp (Wars). 71 (1), 113-128 (2011).</li><li>Heberlein, K. R., 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+novel+mRNA+binding+protein+complex+promotes+localized+plasminogen+activator+inhibitor-1+accumulation+at+the+myoendothelial+junction.">A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction.</a> Arterioscler Thromb Vasc Biol. 32 (5), 1271-1279 (2012).</li><li>Straub, A. 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=Compartmentalized+connexin+43+s-nitrosylation/denitrosylation+regulates+heterocellular+communication+in+the+vessel+wall.">Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.</a> Arterioscler Thromb Vasc Biol. 31 (2), 399-407 (2011).</li><li>Cucina, A., 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=Vascular+endothelial+growth+factor+increases+the+migration+and+proliferation+of+smooth+muscle+cells+through+the+mediation+of+growth+factors+released+by+endothelial+cells.">Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells.</a> J Surg Res. 109 (1), 16-23 (2003).</li><li>Heberlein, K. R., 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=Plasminogen+activator+inhibitor-1+regulates+myoendothelial+junction+formation.">Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.</a> Circ Res. 106 (6), 1092-1102 (2010).</li><li>Isakson, B. E., Best, A. K., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+protein+on+actin+bridges+between+endothelium+and+smooth+muscle+in+arterioles+demonstrates+heterogeneous+connexin+expression+and+phosphorylation.">Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2898-H2904 (2008).</li><li>Biwer, L. A., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-mediated+signalling+in+cellular+microdomains.">Endoplasmic reticulum-mediated signalling in cellular microdomains.</a> Acta Physiol (Oxf). 219 (1), 162-175 (2017).</li></ol>

The VCCC has a number of advantages in terms of recapitulating MEJs in vitro, but there are points of discussion when determining if the VCCC can be utilized for specific application. For example, if different cell types are to be used with this model, it will need to be optimized. We have used primary human cells but it is possible to isolate cells from bovine aortas24, or wildtype or knockout mice, and use them in the VCCC1,5<su...

In small diameter resistance arteries, a thin internal elastic lamina (IEL) separates endothelial (EC) and smooth muscle cells (SMC). The cells can project through holes in this elastic matrix and make direct cytoplasmic connections via gap junction channels1,2. This unique structure is termed the myoendothelial junction (MEJ). The MEJ is a small (approximately 0.5 &#181;m x 0.5 &#181;m, depending on the vascular bed), cellular projection composed predominantly of endothelial cells but may originate from smooth muscle as well3,4. A number of investigators have demonstrated the immense complexity of signaling networks that occur selectively at the MEJ, rendering it an especially important location for facilitating bi-directional signaling between endothelium and smooth muscle5,6,7,8,9,10.
However, a mechanistic dissection of signaling pathways at the MEJ is difficult in an intact artery. Because the MEJ is a cellular projection, it is not currently possible to isolate an in vivo MEJ from the vascular wall. For this reason, the VCCC model1 was developed. Importantly, the VCCC replicates physiological endothelial morphology11 and polarization of signaling between the apical and MEJ portions of the cell12. It was this unique model that facilitated the discovery that alpha hemoglobin is in the endothelial cell, polarized to the MEJ. This is in contrast to conventionally cultured endothelial cells which do not express alpha hemoglobin13. For researchers interested in microvascular endothelium, it may be more appropriate to use endothelial cells that have been cultured in the VCCC, particularly if the intent is to dissect signaling pathways that occur in small diameter resistance arteries.
Using a sturdy plastic insert containing a filter with small diameter pores (0.4 &#181;m in diameter) to co-culture two distinct cell types prevents the cells from migrating between layers. It results in a 10 &#181;m distance between the cells, which is significantly longer than in vivo, but still replicates many of the in vivo characteristics of MEJs, including protein localization and second messenger signaling1,14. In addition, the VCCC allows for targeting of cell type-specific signaling via the addition of an agonist or antagonist to the specific cellular compartment. For example, loading the EC with BAPTA-AM to chelate calcium, and stimulating the SMC with phenylephrine14. In contrast to other descriptions of co-culture models15,16,17,18,19,20,21, this provides instructions on isolating the distinct fractions for mRNA and protein, including the distinct MEJ fraction within the filter pores. The addition of this technique to the VCCC allows for specific investigation of changes in mRNA localization or transcription22, protein phosphorylation12,23, and protein activity12. This article will describe plating of EC on top of the filter and SMC on the bottom, although it is possible to culture the two cell types in different conformations11,18,24.

<table>
	<tbody>
		<tr>
			<td>24mm diameter, 0.4&#181;m Transwell Polyester membrane filter insert</td>
			<td>Corning</td>
			<td>3450</td>
			<td>This is one 6 well plate of inserts</td>
		</tr>
		<tr>
			<td>225cm2 flask</td>
			<td>Corning</td>
			<td>431082</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates (without filter inserts)</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery endothelial cells</td>
			<td>Lonza</td>
			<td>CC-2585</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery smooth muscle cells</td>
			<td>Lonza</td>
			<td>CC-2583</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 EC Basal media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EC growth factors (EGM2 MV Single quots)</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>SmBM SMC basal media</td>
			<td>Lonza</td>
			<td>CC-3181</td>
			<td></td>
		</tr>
		<tr>
			<td>SMC growth factors (SmGM-2 SingleQuot)</td>
			<td>Lonza</td>
			<td>CC-4149</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA 10X</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>Dilute to 2X with sterile dPBS</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>large petri dishes for SMC Plating (150mm)</td>
			<td>Corning</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine gelatin</td>
			<td>Sigma</td>
			<td>G-9382</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td>5&#181;g/ml of fibronectin and store at -20</td>
		</tr>
		<tr>
			<td>10X Hanks&#39; Buffered salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>10X Dulbecco&#39;s Phosphate buffered saline (dPBS)</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>Disposable Curved Scalpel (30mm cutting edge) with handle</td>
			<td>Amazon</td>
			<td>MDS15210</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher</td>
			<td>17-467-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>BD Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL conical tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>10mm petri dishes</td>
			<td>Falcon</td>
			<td>35-1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA lysis reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>Make 4% solution with dPBS</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>04-355-305</td>
			<td></td>
		</tr>
		<tr>
			<td>Cavicide disinfectant</td>
			<td>Fisher</td>
			<td>131000</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA protein lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic dismembranator</td>
			<td>Fisher</td>
			<td>Model 50</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS for harvesting EC/SMC and immunocytochemistry</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>does not need to be sterile</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Embedding/Sectioning/Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Fisher</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated tissue processer</td>
			<td>Excelsior</td>
			<td>ES</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding station + cold plate</td>
			<td>Leica</td>
			<td>HistoCore Arcadia</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek Accu-Edge Microtome blade</td>
			<td>VWR</td>
			<td>25608-961 or 25608-964</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher</td>
			<td>22-230-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermo Fisher</td>
			<td>12-548-5E</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold mounting medium</td>
			<td>Thermo Fisher</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other Solutions</td>
			<td></td>
			<td></td>
			<td>**Perform all steps in sterile conditions if using the solution on live cells</td>
		</tr>
		<tr>
			<td>Sterile dPBS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>sterile HBSS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>Fibronectin stock solution</td>
			<td></td>
			<td></td>
			<td>Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed 
			Final concentration: 100&#181;g/mL</td>
		</tr>
		<tr>
			<td>Fibronectin to coat SMC side of VCCC</td>
			<td></td>
			<td></td>
			<td>thaw one aliquot and add 1mL of fibronectin (100&#181;g/mL) to 19mLs of 1X HBSS to reach a final concentration of 5&#181;g/mL. 
			Final concentration: 5&#181;g/mL</td>
		</tr>
		<tr>
			<td>2x Trypsin EDTA solution</td>
			<td></td>
			<td></td>
			<td>Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) . 
			Final concentration: 2X</td>
		</tr>
		<tr>
			<td>Blocking/antibody solution</td>
			<td></td>
			<td></td>
			<td>(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)</td>
		</tr>
		<tr>
			<td>Bovine gelatin to coat EC VCCC</td>
			<td></td>
			<td></td>
			<td>Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions. 
			Final concentration: 0.50%</td>
		</tr>
		<tr>
			<td>1X PBS for harvesting cells</td>
			<td></td>
			<td></td>
			<td>does not need to be sterile</td>
		</tr>
	</tbody>
</table>

a cell culture model of resistance arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

The filter inserts used for the VCCC have small, 0.4 &#181;m holes. Figure 1A, an en face view of the VCCC filter inset, shows the pores in which the MEJ can form in vitro. The schematic depicts the smooth muscle cells plated on the lower side of the filter one day before the endothelial cells are plated on the opposite side (Figure 1B). Once the cells are plated, the EC, MEJ, and SMC fractions can be isolated t...

Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

A Cell Culture Model of Resistance Arteries

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

The overall goal of this technique is to culture endothelial and smooth muscle cells together in-vitro to create myoendothelial junctions that occur in small diameter resistance arteries. This method can help answer key questions in the cardiovascular field such as, protein localization and activation of signaling cascades in the arterial wall. The main advantage of this technique is that you can specifically isolate myoendothelial junction fractions from the endothelium and smooth muscle, which is impossible to do using an actual artery.Visual demonstration of a paraffin embedding is particularly critical, as the embedding steps can be difficult to learn without seeing the process. Begin construction of the vascular cell co-culture or VCCC, by spraying 150 millimeter petri dishes and lids with disinfectant and then wiping with a paper towel or lint-free wipes. Then, spray the petri dishes with 70%ethanol and place them in the hood to air dry.Open the plate of filter inserts under sterile conditions. Coat the bottom side of the filters with fiberactin solution by pipetting up to one milliliter of fiberactin solution through the side slits into the bottom of the plate. Make sure, that the bottom half of the filter is covered and leave the inserts in the hood, keeping the plate cover.After 30 minutes of treatment with fiberaction solution, vacuum any excess solution without disturbing the filter inserts. Then, invert the plate, placing the filters into the bottom half of the clean petri dish. Trypsinize a 225 square centimeter flask of smooth muscle cells with three milliliters of pre-warmed trypsin EDTA solution.Once the cells have lifted off, add nine milliliters of SMC medium to neutralize the trypsin. Transfer to a conical tube and mix well. Then, pipette 10 microliters onto a hemocytometer to determine cell number.After performing a cell count, carefully plate 750 microliters of the cell suspension containing approximately 75, 000 smooth muscle cells onto the bottom side of each filter. Incubate the plate at 37 degree celsius overnight. On day two, fill each well of a clean six well plate with two milliliters of fresh pre-warmed SMC medium.Remove the medium from an insert by suction and transfer it to the six well plate with SMC medium. Add one milliliter of a 0.5%bovine gelatin solution to the upper side of the inserts and place at 37 degree celsius, for at least 30 minutes. Trypsinize a 225 square centimeter flask of endothelial cells with three milliliters of pre-warmed trypsin EDTA solution.With gentle tapping of the flask to lift the cells off the plate. After re-suspending the cells in EC medium and performing a cell count, remove the gelatin from the filter. Then, plate 360, 000 ECs in a one milliliter volume onto the top side of each filter insert.Let the cells incubate undisturbed at 37 degree celsius for 24 hours. Begin fractionation, by suctioning off the medium from one six well plate of inserts in the cell culture hood and then, transport to a cold room on ice. In the cold room, pipette 10 microliters of PBS onto the SMC side of the inserts.Use the cell lifter to scrape down the SMCs. Then, transfer the cells from the scrapper to a labeled petri dish containing lysis buffer. Once the cell scrapper has touched the lysis buffer, do not allow it to touch the insert filter until it has been completely wiped and dried off on paper towels.Repeat the scrapping of the SMC filter once or twice more, to fully remove the remaining SMC cell debris. Next, pipette 10 microliters of PBS onto the endothelial cell side of the insert filter. Scrape the cells into an appropriately labeled petri dish of lysis buffer, as just demonstrated.Collect the cell slurry from the EC side of the filter with the pipette and transfer to an appropriately labeled petri dish. Be very thorough in scrapping the cells off both sides of the filters to ensure minimal cell contamination in the myoendothelial junction fraction. Next, carefully use the scalpel to cut out the filters from the plastic insert.Do this by cutting 70 to 80%of it away from the plastic and using forceps to pull the filter completely off the plastic insert structure. Point each filter into a labeled 50 milliliter conical tube containing lysis buffer. Ensure that the filters are immersed in buffer and remain wet.After all the cells have been harvested from the filters, vortex the 50 milliliter MEJ tube on full strength for 15 seconds or until mixed well. Remove the filters from the MEJ conical with forceps, dragging them along the side walls of the tube as to leave the maximum amount of proteins and liquids in the tube. Once all the filters have been removed from the MEJ conical tube, do a quick spin in a centrifuge to pull all the liquid and proteins to the bottom of the tube.After the spin, transfer the contents of the MEJ tube and the SMC and EC petri dishes into separate smaller centrifuge tubes. Then, centrifuge the tubes at close to 16, 000 x G for 15 minutes at four degree celsius. Remove the supernatants.Then, assay cell lysates for protein content using a byzinconetic assay. On day five of VCCC incubation, add 4%PFA to each well containing a rinsed filter insert and incubate overnight with shaking at four degree celsius. After approximately 24 hours, transfer the inserts to 70%ethanol for at least 24 hours.Process the filters on a long run on an automated tissue processor. After the run, remove a filter from its cassette using forceps to hold the filter at the edge and then, cut the filter in half with scissors. Lay each of the two halves on a cold plate and fill the embedding mold with liquid paraffin.Very briefly touch the filled embedding mold to the cold plate, then embed each half of the filter into the embedding mold with the cut side down to ensure that the filter is sectioned from the center. Use forceps to hold the filter in place to prevent the halves from falling into each other until the paraffin is cool enough for them to stand alone. Then, move the embedding mold to a cold plate until completely solidify.Embed and section the filtered insert in a vertical orientation. After sectioning, the VCCC can be immunostained for transverse immunofluorescence. This immunotransmission electron microscope image shows a mouse artery with expression of alpha globin at the MEJ labeled with gold beads, which appear as black dots.This western blot demonstrates that plasminogen activator inhibitor one expression is enriched in the MEJ fraction versus the EC or SMC fractions. Plastic EC indicates ECs grown on a plastic dish which do not form MEJ. Immunostaining reveals the tight compartmentalization of the VCCC model.The alpha-1 adrenergic receptor one is seen only in the smooth muscle cell layer, the bradykinin receptor is seen only in the endothelial cell layer. F actin staining is throughout both cell types at the in-vitro MEJ as indicated by the white arrow. While attempting this procedure, it is important to remember to keep everything sterile until the time of harvest, carefully plate the cells and thoroughly scrape the filters.Following this procedure, other methods like western blotting or immunofluorescence can be performed in order to answer additional questions lik protein localization, expression levels or activation.

a-cell-culture-model-of-resistance-arteries

Research

JoVE Journal

Medicine

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.