Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Summary

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.

Abstract

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 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 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.

Introduction

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 response^3,4. To address this issue, an ex 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 “arterialized vein” 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 IH^6-10. 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 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.

Protocol

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

1. 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.
2. Make a median incision from the groin to the knee (leaving the interrupted skin portion).
3. 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 °C in a RPMI-1640 Glutamax medium, supplemented with 12.5% fetal calf serum and bring it to the laboratory.

2. EVPS Design

1. 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.
2. 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.
3. 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.
4. Design the pressure column, such that the “resting pressure” applied to the system is: p = 0-10 = h x ρ x g, where p = pressure (N/m², Pa) h = height of fluid column (m) ρ = 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.
5. Prepare the medium. Based on previous studies^5,11-14, 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 μg/ml: gentamycin). Shear stress (SS) is given by SS= 4 μQ/π*r³Q is the flow rate (ml/sec), r the radius (cm) of the vein segment, and μ is the viscosity of the perfusion medium.
   1. 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.
6. Set the gearing pump to induce a pulsatile cardioid signal of 60 pulses/min and constant amplitude generating a unidirectional flow of 150 ± 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.

3. EVPS Assembly (Figure 1)

1. Before starting, make sure all the equipment is sterile. Perform all the following steps under asepsis in a laminar flow hood.
2. Place the vein in a Petri dish filled with medium. Use a surgical blade and divide the vein into 3 equal segments.
3. 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.
4. Use the 2 remaining segments for perfusion.
   1. Very gently inject medium into the vein and determine the normal flow direction; in presence of valves the vein is reversed.
   2. Sealing the veins is of utmost importance to experimental success. Check for leaks through collaterals. Secure any leaks with 6-0 silk sutures.
5. 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).
   1. 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.
6. To reinforce (mesh) the second segment, release the two cylinders (with the vein attached) from the L-shaped pieces (2.3 and Figure 1).
   1. 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.
   2. Once the mesh covers the entire surface of the vein secure the jacketed vein to the cylinders with Ethibon 3-0.
   3. Reassemble the vein/cylinder compound to the L-shaped support and transfer it to the perfusion chamber, previously filled with medium.
7. Connect each metallic cylinder (in-and outflow) to a Y-splitter using peroxide-treated silicone tubing with an internal diameter of 3.2 mm.
8. 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).
9. Inside the incubator, use a long (one-meter length) tube to connect the pressure column to the pump head.
10. Complete the set up by connecting the pump head to the inflow Y-splitter with another long length tube (Figure 1).

4. Veins Perfusion

1. 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 ± 0.1 °C with a pH kept constant at 7.40 ± 0.01 (using a CO₂/pH algorithm based on the Henderson-Hasselbach equation).
2. Bring the gear pump head outside the incubator and connect it to the gear pump drive. Screw the rods to secure the assembly.
3. 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.
4. 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.
   1. Make sure the tube is entirely filled with medium and does not contain any bubbles. De-bubble the culture system through the “arterial line” 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 < 0 and the column progressively empties look for a leak (vein collateral or inadequate seal between the vein and the tube).
5. 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.
6. 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.

5. Completion of the Perfusion

1. 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.

Results

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...

Discussion

This study uncovers an ex 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 grafts^5,11,12,15.

Reproducible and quantifiable hem...

Materials

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