This work was supported by grants from the SNF [31003A-138528], the Octav and the Marcella Botnar Foundation, the Novartis Foundation and the Emma Muschamp Foundation. We thank Martine Lambelet, and&#160;Jean-Christophe Stehle for their excellent technical assistance.

The Ethical Committee of the University of Lausanne approved the experiments, which are in accordance with the principles outlined in the Declaration of Helsinki of 1975, as revised in 1983 for the use of human tissues.
1. Human Great Saphenous Vein Harvest
<ol>
	<li>Obtain surplus segments of non-varicose human saphenous veins from patients undergoing lower limb bypass surgery for ischemia. In the operating room, disinfect the entire leg with an iodine solution and drape the patient to expose the leg from the groin to the foot.</li>
	<li>Make a median incision from the groin to the knee (leaving the interrupted skin portion).</li>
	<li>Harvest the great saphenous vein with a pedicle of surrounding tissue (no-touch technique). Secure side branches of the veins with 4-0 silk ties. Immediately store a minimum of 9 cm long surplus segment of the greater saphenous vein, with an external diameter of 2.5-4 mm at 4 &#176;C in a RPMI-1640 Glutamax medium, supplemented with 12.5% fetal calf serum and bring it to the laboratory.</li>
</ol>
2. EVPS Design
<ol>
	<li>Assemble the general equipment shown in Figure 1. Autoclave all equipment and keep all components under sterile conditions. In addition, ensure that the system is waterproof and does not leak chemicals into the medium. Use polymethacrylate methyl (PMMA-GS) for the cover. Steel (X5 Cr Ni 18 10) and polyoxymethylene plastic (POM) as the vein support.</li>
	<li>Design the perfusion chamber to the desired geometry to allow the placement of the vein and its connection. Make sure the depth (or radius if using cylindrical construction) is at least 2.5 cm so it allows minimal flexion and dilatation of the vessel along with constant coverage by the culture media (Figure 1). Sealing is a major issue and is the reason rectangular PMMA-GS construction is used.</li>
	<li>Design the vein support to the desired geometry. To avoid vein kinking or over distension, allow length adjustment by pushing or pulling (screw cannot be used to that purpose, as the vein would be twisted along with the screw). 
	NOTE: A full steel rod connected by 2 sliding L-shaped pieces that support the 2 vein cylinders (5 mm diameter to fit the vessel) and the vein (Figure 1B and Figure 2) is used here.</li>
	<li>Design the pressure column, such that the &#8220;resting pressure&#8221; applied to the system is: p = 0-10 = h x &#961; x g, where p = pressure (N/m2, Pa) h = height of fluid column (m) &#961; = density of liquid (kg/m3) and g = the gravitational constant (9.81 m/sec2). Design four connection ducts, from top to bottom: to apply pressure, for the outflow (from the vein), the inflow (to the vein) and to allow medium change.</li>
	<li>Prepare the medium. Based on previous studies5,11-14, choose RPMI-1640, supplemented with Glutamax, 12.5% fetal calf serum, and 1% antibiotic-antimycotic solution (10,000 U/ml penicillin G, plus 10 mg/ml streptomycin sulfate, plus 25 mg/ml amphotericin B, plus 0.5 &#956;g/ml: gentamycin). Shear stress (SS) is given by SS= 4 &#956;Q/&#960;*r3Q is the flow rate (ml/sec), r the radius (cm) of the vein segment, and &#956; is the viscosity of the perfusion medium.
	<ol>
		<li>Modulate SS by adjusting the viscosity through addition of 70 kDa dextran. Measure the viscosity with a viscometer. Here, add 8% 70 kDa dextran to set SS to 9-15 dyn/cm2.</li>
	</ol>
	</li>
	<li>Set the gearing pump to induce a pulsatile cardioid signal of 60 pulses/min and constant amplitude generating a unidirectional flow of 150 &#177; 15 ml/min, independent from the pressure applied in the system and controlled by a computer. Ensure that the driving software integrates constant acquisition and monitoring of pressures, flow velocity, pulse rate, and signal. If desired, use a second pump (non synchronized) to produce a non-laminar, turbulent flow.</li>
</ol>
3. EVPS Assembly (Figure 1)
<ol>
	<li>Before starting, make sure all the equipment is sterile. Perform all the following steps under asepsis in a laminar flow hood.</li>
	<li>Place the vein in a Petri dish filled with medium. Use a surgical blade and divide the vein into 3 equal segments.</li>
	<li>Immediately rinse one segment in PBS. Divide the segment in 3 parts, fix one in formalin for morphometry. Freeze the other two for quantitative transcript (RT-PCR) and protein (western blot) analysis. Consider these segments as a control, non-perfused vein.</li>
	<li>Use the 2 remaining segments for perfusion.
	<ol>
		<li>Very gently inject medium into the vein and determine the normal flow direction; in presence of valves the vein is reversed.</li>
		<li>Sealing the veins is of utmost importance to experimental success. Check for leaks through collaterals. Secure any leaks with 6-0 silk sutures.</li>
	</ol>
	</li>
	<li>Connect the vein segment between the two metallic cylinders, one end at a time (2.3, Figure 1). Secure the cylinders with Ethibon 3-0 around the indentations (Figure 1A and B).
	<ol>
		<li>Place the entire venous segment into the perfusion chamber previously filled with medium. Repeat the same procedure for the second segment. 
		NOTE: Failure to properly seal the vein to the cylinder will be a source of leak, require reintervention, and significantly increase the risk of infection and experimental failure.</li>
	</ol>
	</li>
	<li>To reinforce (mesh) the second segment, release the two cylinders (with the vein attached) from the L-shaped pieces (2.3 and Figure 1).
	<ol>
		<li>Be gentle and do not touch the vein with any instruments. Slide the mesh first on the cylinder then onto the vein. A push/pull jostling will get the mesh on the vein.</li>
		<li>Once the mesh covers the entire surface of the vein secure the jacketed vein to the cylinders with Ethibon 3-0.</li>
		<li>Reassemble the vein/cylinder compound to the L-shaped support and transfer it to the perfusion chamber, previously filled with medium.</li>
	</ol>
	</li>
	<li>Connect each metallic cylinder (in-and outflow) to a Y-splitter using peroxide-treated silicone tubing with an internal diameter of 3.2 mm.</li>
	<li>Connect the outflow splitter to a second Y-splitter using the same type of tubing. From this Y-splitter, use one tube to measure the perfusion pressure through both vessels. Connect the other one back to the column to form a closed loop system (Figure 2).</li>
	<li>Inside the incubator, use a long (one-meter length) tube to connect the pressure column to the pump head.</li>
	<li>Complete the set up by connecting the pump head to the inflow Y-splitter with another long length tube (Figure 1).</li>
</ol>
4. Veins Perfusion
<ol>
	<li>After the EVPS assembly has been completed, fill the column with medium (stay below the vein outflow duct to allow refilling). Add more medium into the column until the system is full. Move all the system into the incubator maintained at 37 &#177; 0.1 &#176;C with a pH kept constant at 7.40 &#177; 0.01 (using a CO2/pH algorithm based on the Henderson-Hasselbach equation).</li>
	<li>Bring the gear pump head outside the incubator and connect it to the gear pump drive. Screw the rods to secure the assembly.</li>
	<li>Switch on the pump power, make sure it is activated on the driving software and allow 5 min for the medium to be equally distributed in every compartment.</li>
	<li>To monitor the pressure, use an arterial line monitoring. Connect the EVPS pressure output (it corresponds to the arterial catheter) to the pressure transducer linked to the computer.
	<ol>
		<li>Make sure the tube is entirely filled with medium and does not contain any bubbles. De-bubble the culture system through the &#8220;arterial line&#8221; tube (Figure 2). Pay attention to the display and look for a pulsatile cardioid signal of 60 pulses/min of constant amplitude. At this point, the average pressure is between 0-10 mm Hg. If the pressure is &#60; 0 and the column progressively empties look for a leak (vein collateral or inadequate seal between the vein and the tube).</li>
	</ol>
	</li>
	<li>Set the minimal pressure to 6 mm Hg for a venous test or at 90 mm Hg for an arterial test. Under these conditions, an air injector applies the required pressure to the column and system.</li>
	<li>Change the medium every 2 days by using the tube connected to the pressure column. To prevent pressure change damage, open the column plug first.</li>
</ol>
5. Completion of the Perfusion
<ol>
	<li>After 3 or 7 days of perfusion: take the EVPS out of the incubator and dismount the veins. Discard the 5 mm proximal and distal vein ends attached to the equipment. Cut a central, 5 mm thick rings from the remaining segment and fix in formalin (morphometry). Freeze the remaining fragments and reduce into powder for further molecular analyses.</li>
</ol>

<ol>
	<li>Sal Go, 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=Executive+summary:+heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+american+heart+association.">Executive summary: heart disease and stroke statistics--2014 update: a report from the american heart association.</a> Circulation. 129, 399-410 (2014).</li><li>Sal Conte, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+PREVENT+III:+a+multicenter,+randomized+trial+of+edifoligide+for+the+prevention+of+vein+graft+failure+in+lower+extremity+bypass+surgery.">Results of PREVENT III: a multicenter, randomized trial of edifoligide for the prevention of vein graft failure in lower extremity bypass surgery.</a> Journal of Vascular Surgery. 43, 742-751 (2006).</li><li>Yu, P., Nguyen, B. T., Tao, M., Bai, Y., Ozaki, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+vein+graft+hemodynamic+manipulations+to+enhance+experimental+utility.">Mouse vein graft hemodynamic manipulations to enhance experimental utility.</a> The American Journal of Pathology. 178, 2910-2919 (2011).</li><li>Davies, M. G., Hagen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprinted+article+&amp;#34;Pathophysiology+of+vein+graft+failure:+a+review&amp;#34;.">Reprinted article &amp;#34;Pathophysiology of vein graft failure: a review&amp;#34;.</a> European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. 42, S19-S29 (2011).</li><li>Berard, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hemodynamic+forces+in+the+ex+vivo+arterialization+of+human+saphenous+veins.">Role of hemodynamic forces in the ex vivo arterialization of human saphenous veins.</a> Journal of Vascular Surgery. 57, 1371-1382 (2013).</li><li>Vijayan, V., 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=Long-term+reduction+of+medial+and+intimal+thickening+in+porcine+saphenous+vein+grafts+with+a+polyglactin+biodegradable+external+sheath.">Long-term reduction of medial and intimal thickening in porcine saphenous vein grafts with a polyglactin biodegradable external sheath.</a> Journal of Vascular Surgery. 40, 1011-1019 (2004).</li><li>Jeremy, J. Y., 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=On+the+biology+of+saphenous+vein+grafts+fitted+with+external+synthetic+sheaths+and+stents.">On the biology of saphenous vein grafts fitted with external synthetic sheaths and stents.</a> Biomaterials. 28, 895-908 (2007).</li><li>Zilla, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constrictive+external+nitinol+meshes+inhibit+vein+graft+intimal+hyperplasia+in+nonhuman+primates.">Constrictive external nitinol meshes inhibit vein graft intimal hyperplasia in nonhuman primates.</a> The Journal of Thoracic and Cardiovascular Surgery. 136, 717-725 (2008).</li><li>Zilla, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+shape+memory+in+external+vein-graft+meshes+allows+extreme+diameter+constriction+for+suppressing+intimal+hyperplasia:+a+non-human+primate+study.">Utilization of shape memory in external vein-graft meshes allows extreme diameter constriction for suppressing intimal hyperplasia: a non-human primate study.</a> Journal of Vascular Surgery. 49, 1532-1542 (2009).</li><li>Yeoman, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+constitutive+model+for+the+warp-weft+coupled+non-linear+behavior+of+knitted+biomedical+textiles.">A constitutive model for the warp-weft coupled non-linear behavior of knitted biomedical textiles.</a> Biomaterials. 31, 8484-8493 (2010).</li><li>Longchamp, 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=The+use+of+external+mesh+reinforcement+to+reduce+intimal+hyperplasia+and+preserve+the+structure+of+human+saphenous+veins.">The use of external mesh reinforcement to reduce intimal hyperplasia and preserve the structure of human saphenous veins.</a> Biomaterials. 35, 2588-2599 (2014).</li><li>Saucy, 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=Ex+vivo+pulsatile+perfusion+of+human+saphenous+veins+induces+intimal+hyperplasia+and+increased+levels+of+the+plasminogen+activator+inhibitor+1.">Ex vivo pulsatile perfusion of human saphenous veins induces intimal hyperplasia and increased levels of the plasminogen activator inhibitor 1.</a> European Surgical Research. Europaische Chirurgische Forschung. Recherches Chirurgicales Europeennes. 45, 50-59 (2010).</li><li>Dubuis, 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=Atorvastatin-loaded+hydrogel+affects+the+smooth+muscle+cells+of+human+veins.">Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins.</a> The Journal of pharmacology and experimental. 347, 574-581 (2013).</li><li>Deglise, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+connexin43+expression+in+human+saphenous+veins+in+culture+is+associated+with+intimal+hyperplasia.">Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.</a> Journal of Vascular Surgery. 41, 1043-1052 (2005).</li><li>Muto, A., Model, L., Ziegler, K., Eghbalieh, S. D., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+vein+graft+adaptation+to+the+arterial+circulation:+insights+into+the+neointimal+algorithm+and+management+strategies.">Mechanisms of vein graft adaptation to the arterial circulation: insights into the neointimal algorithm and management strategies.</a> Circulation Journal : Official Journal of the Japanese Circulation Society. 74, 1501-1512 (2010).</li><li>Tao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+murine+intimal+hyperplasia+model+founded+on+a+focal+carotid+stenosis.">A simplified murine intimal hyperplasia model founded on a focal carotid stenosis.</a> The American Journal of Pathology. 182, 277-287 (2013).</li><li>Berard, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salvage+treatment+for+venous+aneurysm+complicating+vascular+access+arteriovenous+fistula:+use+of+an+exoprosthesis+to+reinforce+the+vein+after+aneurysmorrhaphy.">Salvage treatment for venous aneurysm complicating vascular access arteriovenous fistula: use of an exoprosthesis to reinforce the vein after aneurysmorrhaphy.</a> European Journal of Vascular and Endovascular Surgery : the Official Journal of the European Society for Vascular Surgery. 40, 100-106 (2010).</li></ol>

The authors have nothing to disclose.

This study uncovers an ex&#160;vivo vein perfusion system (EVPS) to perform extensive hemodynamic studies in human veins. This system allows saphenous veins perfusion under defined hemodynamic parameters in the absence of aggravating inflammatory and growth factors released by circulating cells in vivo. Thus, it provides a better understanding of the underlying pathways involved in the control of IH in human veins grafts5,11,12,15.
Reproducible and quantifiable hem...

Cardiovascular diseases are the leading cause of morbidity and mortality in Western countries1. Despite advances made in endovascular treatments, bypass surgery remains the mainstay of contemporary therapies, thus over half a million vein grafts are performed annually in the United States. However, despite decades of research, 30-60% of lower extremity vein grafts fail within the first years due to intimal hyperplasia (IH)2. Mechanical forces, particularly low shear stress (SS) and high wall tension, are pivotal in the initiation and development of this hyperplastic response3,4. To address this issue, an ex&#160;vivo veins perfusion system (EVPS) was generated to study, under strictly controlled hemodynamic conditions (pressure and shear stress), the behavior of human saphenous veins. In this study, following insertion into the arterial-like circulation, high pressure (mean = 100 mm Hg) was sufficient to stimulate proliferation and migration of smooth muscle cells into the intimal layer (IH)5.
Mammalian studies have suggested the use of external reinforcement as an efficient method to support the &#8220;arterialized vein&#8221; and counteract the acute hemodynamic changes the vein faces once implanted into an arterial milieu. The mesh prevented over-distension, increased shear stress, and reduced wall tension and consequently IH6-10. However, the underlying mechanisms and its applicability to human veins in improving bypass patency have not been fully characterized. Our EVPS was used to compare, in condition mimicking the alterations a vein faces once inserted into an arterial regimen (high shear stress and pressure), the behavior of human saphenous veins in the absence and presence of an external macroporous polyester tubular mesh. By preventing pathological remodeling and IH, the mesh provided evidence of its potential clinical efficiency11.
This study 1) introduces a model of ex&#160;vivo human saphenous veins perfusion under controlled pressure and shear stress 2) demonstrates that external macro-porous polyester mesh reduces IH and provides crucial information for its potential clinical application.

<table border="0" cellpadding="0" cellspacing="0" width="1113">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Name</td>
			<td style="width:371px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:144px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 - Glutamax</td>
			<td>Life Technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"><a name="RANGE!A3">Penicilline/Streptomycine/Fungizone</a></td>
			<td>Bioconcept</td>
			<td>4-02F00-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran from Leuconostoc spp. 500 gr.</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tampon PBS CHUV pH 7.1-7.3 1 lt.</td>
			<td>Laboratorium und Grosse Apotheke Dr. G. Bichsel AG</td>
			<td>100 0 324 00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon Tubing (Peroxide) L/S 16 (96400-16 ) - 7.5m</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MF0037ST</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-splitter&#160;</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>Y-connector</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm Culture dish</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430165-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gearing pump - Reglo-Z</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>SM 895&#160;</td>
			<td>&#160;App-Nr 03736-00194</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump Head</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MI0008&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring Kit TRANSPAC IV</td>
			<td>icumedical</td>
			<td>011-0J736-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 mL Syringes</td>
			<td>B. Braun Medical SA</td>
			<td>4612041-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Etibon 3-0 FS-2</td>
			<td>Ethicon- Johnson&#38;Johnson</td>
			<td>EH7346H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mesh ProVena 6-8mm</td>
			<td>B. Braun Medical SA</td>
			<td>1105012-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl: Sodium Chlorure Solution perfusion 0.9% (100 ml)</td>
			<td>B. Braun Medical SA</td>
			<td>534534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Masterflex L/S Standard Drive</td>
			<td>Cole-Parmer Instrument Co</td>
			<td>7521-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition card</td>
			<td>National Instruments</td>
			<td>PCI-6024 E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowmeter module</td>
			<td>Transonic Systems Inc.</td>
			<td>TS410 and T402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stopcock with 3-ways</td>
			<td>BD Connexta Luerlock</td>
			<td>394600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex Filter</td>
			<td>Milian</td>
			<td>SE2M229I04</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for human saphenous veins ex vivo perfusion and external reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

The EVPS provides a valuable tool to independently assess the hemodynamic forces on human saphenous vein grafts remodeling and IH.
Figure 1 shows the perfusion chamber and the vein support. In Figures 1A and B, the vein support before (Figure 1A) and after (Figure 1B) assembly, respectively, is pictured. It is composed (from the top to the bottom) of 1 plain stainless steel tube measuring 9 cm, which serves as...

Watch this Scientific Journal Video about Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement at JoVE.com

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

procedure-for-human-saphenous-veins-ex-vivo-perfusion-external

Research

JoVE Journal

Medicine

11.1K Views.  Brigham and Women's Hospital/Harvard Medical School. The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

Video: Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

External Cephalic Version: Is it an Effective and Safe Procedure?

External cephalic version (ECV) is an effective procedure for reducing the number of cesarean sections. To date, there is no video publication showing the methodology of this procedure. The main objective is to show how to perform ECV with a specific protocol with tocolysis before the procedure and analgesia. Moreover, we describe and analyze the factors associated with successful ECV, and also compare to deliveries in the general pregnant population.
A retrospective and descriptive analysis of ECV carried out at the Hospital Clinico Universitario Virgen de la Arrixaca in Murcia (Spain) between 1/1/2014 and 12/31/2018 was assessed. The latest data available of labor deliveries in the local center, which is the biggest maternity department in Spain, were from 2018.
320 patients were recruited and 3 pregnant women were lost during the study. ECV was carried out at 37&#177;3 weeks gestation. ECV was successful in 82.5% (N=264). 19 complications were reported (5.9%): 8 vaginal bleeding (2.5%), 9 fetal bradycardia (2.8%), 1 preterm rupture of membranes (0.3%) and 1 cord prolapse (0.3%). A previous vaginal delivery increases the success rate of ECV ORadjusted=3.03 (1.62-5.68). Maternal Body Mass Index (BMI) affects the success of ECV ORadjusted=0.94 (0.89-0.99). Patients with BMI&#62;40 kg/m2 have an ORadjusted=0.09 (0.009-0.89) compared with those with BMI &#60;25 kg/m2. If ECV was successful, the cesarean delivery index is 22.2% (17.5-27.6%), the eutocic delivery index is 52.1% (46.1-58.1%) and the instrumented vaginal delivery index is 25.7% (20.7-31.2%). There are no differences in cesarean and eutocic delivery indexes after successful ECV. However, a successful ECV is associated with a 6.29% increase in the instrumented delivery rate (OR=1.63).
ECV is an effective procedure to reduce the number of cesarean sections for breech presentations. Maternal BMI and previous vaginal delivery are associated with ECV success. Successful ECV does not modify the usual delivery pattern.

This article shows how to perform the external cephalic version (ECV) by two experienced obstetricians in the obstetric operating room in the presence of an anesthesiologist and a midwife. The ECV is carried out with analgesia and tocolysis. Two attempts are made under ultrasonography control.

External cephalic version (ECV) is an effective procedure for reducing the number of cesarean sections. To date, there is no video publication showing the ...

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. 
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. 
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Advancing Lung Transplantation with PolyhHb-Based Perfusion in Rat EVLP Models

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. However, new and exciting technology, such as ex vivo lung perfusion (EVLP), offers lung transplant providers extended assessment for marginal donor allografts. This dynamic assessment platform has led to an increase in lung transplantation and has allowed providers to use donors that were previously discarded, thus expanding the donor pool. Current perfusion techniques use cellular or acellular perfusates, and both have distinct advantages and disadvantages. Perfusion composition is critical to maintaining a homeostatic environment, providing adequate metabolic support, decreasing inflammation and cellular death, and ultimately improving organ function. Perfusion solutions must contain sufficient protein concentration to maintain appropriate oncotic pressure. However, current perfusion solutions often lead to fluid extravasation through the pulmonary endothelium, resulting in inadvertent pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis. Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as the proper protocols to test them in clinically relevant translational transplant models.

Author Spotlight: Utilizing Next-Generation Polymerized Human Hemoglobin for Improved Donor Lung Evaluation and Preservation in Rats

Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat ex vivo lung perfusion.

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. ...

Surgical Technique for Jugular Vein Thermodilution Catheter Placement in Pigs

Determination of Cardiac Output in a Porcine Model for Ex Vivo Pulmonary Perfusion

Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that perfuses lungs that are not suitable for transplantation via an extracorporeal circulation pump to improve their function and increase their viability. Existing EVLP protocols are differentiated by the type of perfusion solution and perfusion flow, which varies from 40%-100% of the estimated cardiac output (CO) according to the body surface area (BSA). Devices for measuring CO use simple physical principles and other mathematical models. Thermodilution in animal models continues to be the reference standard for estimating CO because of its simplicity and ease of reproduction. Therefore, the objective of this study was to reproduce the measurement of CO by thermodilution in pigs and compare its precision and accuracy with those obtained by the BSA, weight, and Fick's method, to establish perfusion flow during EVLP. In 23 pigs, a thermodilution catheter was placed in the right jugular vein, and the carotid artery on the same side was cannulated. Blood samples were obtained for gasometry, and CO was estimated by thermodilution, adjusted body surface area, Fick's principle, and per body weight. The CO obtained by the BSA was greater (p = 0.0001, ANOVA, Tukey) than that obtained by the other methods. We conclude that although the methods used in this study to estimate CO are reliable, there are significant differences between them; therefore, each method must be evaluated by the investigator to determine which meets the needs of the protocol.

Author Spotlight: Assessment of Cardiac Output Calculation by Thermodilution in Pigs for Effective Perfusion Flow During EVLP

This protocol describes the surgical technique used for the placement of a thermodilution catheter through the jugular vein in pigs to estimate cardiac output and ensure adequate lung perfusion during ex vivo lung perfusion (EVLP).

Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that ...