JEK is supported by grants from Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo [FAPESP-INCT-20214/50889-7 and 2013/17368-0] and Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico-CNPq (INCT-465586/2014-7 and 309179/2013-0). AAM is supported by grants from Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP 2015/11139-5) and Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico-CNPq (Universal - 407911/2021-9).

Unused segments of saphenous veins were obtained from patients undergoing aortocoronary bypass surgery at the Heart Institute (InCor), University of S&#227;o Paulo Medical School. All individuals gave informed consent to participate in the study, which was reviewed and approved by the local ethics committee.
1. Isolation, culture, and characterization of primary human saphenous vein endothelial cells (hSVECs)
<ol>
	<li>Preparation
	<ol>
		<li>Autoclave a pair of straight or curved forceps and tissue scissors (7-8 cm).</li>
		<li>Prepare sterile gelatin. Mix 0.1 g (0.1% w/v) or 3 g (3% w/v) of porcine skin gelatin with 100 mL of ultrapure water, autoclave for 15 min, and store at 4 &#176;C. Warm to 37 &#176;C to liquefy before coating the cell culture dishes and slides.</li>
		<li>Prepare sterile collagenase (1 mg/mL) inside the laminar flow hood. Dilute the collagenase in PBS and sterilize it with a 0.2 &#181;m filter.</li>
		<li>Coat a 60 mm cell culture dish and an 8-well chamber slide with 0.1% w/v gelatin for at least 30 min at 37 &#176;C. Remove the excess gelatin prior to seeding the cells.</li>
		<li>Prepare complete endothelial cell medium: endothelial cell growth basal medium (EBM-2) supplemented with EGM2 growth factors. 
		NOTE: The EGM2 growth factors is supplied as a kit by a company listed in the Table of Materials. The concentration of the factors is not disclosed by the company.</li>
		<li>Make 2% and 5% bovine serum albumin (BSA), 4% paraformaldehyde (PFA), and 0.1% Triton X-100 solutions, all in phosphate-buffered saline (PBS).</li>
	</ol>
	</li>
	<li>Isolation and culture of hSVECs
	<ol>
		<li>Collect at least a 2-3 cm long saphenous vein segment for this procedure. 
		NOTE. All the cell culture procedures must be carried out under sterile conditions and inside a laminar flow hood.</li>
		<li>Transfer the vein segment to a Petri dish filled with pre-warmed PBS. Insert a pipette tip into one end of the vein and gently flush with PBS to remove all the blood inside the lumen. Flush it until the washing flow becomes completely clear.</li>
		<li>Close one of the ends of the vein with a sterile cotton suture. Carefully fill the vessel with 1 mg/mL collagenase type II solution. Close the other end of the vessel with a cotton suture (Figure 1A). Incubate the vessel with collagenase solution in a humidified atmosphere of 5% CO2 at 37 &#176;C for 1 h.</li>
		<li>After that, cut one of the ends of the vessel inside a 15 mL tube and collect the collagenase solution. Cut the other end and flush the luminal surface with 1-2 mL of PBS to collect all the detached ECs. Put all the PBS together with collagenase solution inside the same tube.</li>
		<li>Centrifuge the tube at 400 x g for 5 min at room temperature (RT) to pellet the cells. 
		&#8203;NOTE. The cell pellet is very small and difficult to visualize. If the blood is not completely removed before collagenase treatment (step 1.2.2.), the blood cells will also precipitate, and the pellet will be reddish.</li>
		<li>Remove the supernatant and resuspend the cell pellet in 3-4 mL of complete endothelial cell medium (EBM-2 supplemented with EGM2 growth factors) and an additional heparin solution (5 U/mL, final concentration). Plate the cells in a 60 mm cell culture dish pre-coated with 3% w/v gelatin. 
		NOTE: Gelatin was the first choice due to its ease of accessibility and low cost. As the coating with gelatin successfully maintains the EC phenotype, no other coating substance was tested. Collagens and fibronectin can be tested in studies aiming to investigate the influence of different extracellular matrix components on EC adhesion and the phenotype.</li>
		<li>Incubate the cells at 37 &#176;C in a humidified atmosphere with 5% CO2 for 4 days without changing the medium. After that, change the media every other day. From this moment on, the additional cell media supplementation with heparin is no longer necessary. Examine the plate under the microscope to ensure that the red blood cells were removed.</li>
		<li>After 3-10&#160;days post-extraction, depending on the size and diameter of the vein segment, visualize the hSVECs by phase-contrast microscopy.</li>
		<li>Evaluate the degree of confluence and their cobblestone morphology in phase-contrast microscopy.</li>
		<li>Continue changing the media every 2 days until the cells reach 70%-80% confluency.</li>
		<li>Passage the hSVECs with 0.25% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA) solution.
		<ol>
			<li>Wash the cells with pre-warmed PBS.</li>
			<li>Add 1 mL of trypsin-EDTA solution to the 60 mm cell culture dish. Incubate at 37 &#176;C for 2 min or until all the cells are detached.</li>
			<li>Add 2 mL of complete endothelial cell medium to inactivate the trypsin.</li>
			<li>Transfer the cell suspension to a 15 mL tube and centrifuge at 300 x g for 3 min at RT.</li>
			<li>Discard the supernatant and resuspend the cells in 1 mL of culture medium.</li>
			<li>Count the cell suspension in 1:1 trypan blue (0.4%) to plate viable cells.</li>
			<li>To expand the culture, plate the cells in a 100 mm cell culture dish with 10 mL of pre-warmed complete endothelial cell medium and culture it at 37 &#176;C in a humidified atmosphere with 5% CO2. 
			NOTE. On average, the seeding of 4 x 104 hSVEC/cm2 will be 100% confluent after 48 h.</li>
		</ol>
		</li>
		<li>To cryopreserve the cells, resuspend the pellet from step 1.2.11.5 in 5% dimethyl sulfoxide (DMSO) in fetal bovine serum (FBS) and store in liquid nitrogen.</li>
	</ol>
	</li>
	<li>Characterization of hSVECs: Flow cytometry staining for CD31
	<ol>
		<li>Transfer 1 x 105 hSVECs to one 1.5 mL tube and centrifuge at 300 x g for 3 min at RT. Discard the supernatant and resuspend the pellet in 100 &#181;L of PBS containing 2% BSA and CD31-FITC monoclonal antibody (1:100).</li>
		<li>Stain the cells for 30 min at RT in the dark.</li>
		<li>Wash the stained cells by adding 0.5 mL of PBS and centrifuge them at 300 x g for 3 min at RT. Discard the supernatant and resuspend the cell pellet in 400 &#181;L of PBS with 2% BSA.</li>
		<li>Use unlabeled hSVECs, without CD31 antibody, as the negative control.</li>
		<li>Proceed to flow cytometer acquisition analysis using a blue laser and FITC channel (excitation/emission max [Ex/Em] of 498/517 nm). If other fluorochromes are used, check whether the cytometer has appropriate filter sets for their detection. Separate the cells from debris in the function of their size and granularity, then gate single cells by forward scatter and side scatter.</li>
	</ol>
	</li>
	<li>Characterization of hSVECs: Fluorescent staining for CD31 and VE-cadherin
	<ol>
		<li>Plate 0.1 x 105 cells in the gelatin-coated 8-well chamber slide. Incubate the cells overnight at 37 &#176;C, 5 % CO2, to allow the cells to attach to the culture surface.</li>
		<li>Remove the medium and wash the cells once with PBS.</li>
		<li>Add 0.3 mL/well of 4% PFA to fix the cells. Incubate for 15 min at RT.</li>
		<li>Wash three times with PBS.</li>
		<li>Add 0.3 mL/well of 0.1% Triton X-100 solution to permeabilize the cells. Incubate for 15 min at RT.</li>
		<li>Wash three times with PBS.</li>
		<li>To block non-specific antibody binding sites, add 0.3 mL/well of 5% BSA solution to the cells. Incubate for 15 min at RT.</li>
		<li>Wash three times with PBS.</li>
		<li>Incubate the cells with the primary antibodies (CD31 and VE-cadherin, 1:100) diluted in PBS with 2% BSA overnight with gentle rocking at 4 &#176;C. Leave one well incubating with PBS with 2% BSA (no primary antibody) as the negative control.</li>
		<li>Wash three times with PBS.</li>
		<li>Incubate the cells with fluorescently labeled secondary antibodies diluted (1:500) in PBS for 1 h at RT. Add 4&#39;,6-diamidino-2-phenylindole (DAPI) for nuclear staining to a final concentration of 1 mg/mL.</li>
		<li>Wash three times with PBS.</li>
		<li>Remove the media chamber with the separator. Place one drop of the mounting medium reagent (50% glycerol in PBS) and place a glass slide (24 mm x 60 mm) while avoiding trapping bubbles.</li>
		<li>Observe the cells under a fluorescence microscope. 
		&#8203;NOTE: DAPI is detected with a DAPI light cube (Ex: 335-379 nm; Em: 417-477 nm), and CD31/VE-cadherin is detected using a green fluorescent protein (GFP) light cube (Ex: 459-481 nm; Em: 500-550 nm). Light cubes with a similar excitation/emission wavelength range are also adequate for these fluorochromes. If other fluorochromes are used, check whether the microscope used has appropriate filter sets for their detection.</li>
	</ol>
	</li>
</ol>
2. Shear stress on hSVECs
<ol>
	<li>Preparation
	<ol>
		<li>Coat the flow chamber slide with 0.1% w/v gelatin for at least 30 min at 37 &#176;C. Remove excess gelatin prior to seeding the cells.</li>
		<li>Equilibrate the fluidic unit and sterile perfusion set with 10 mL reservoirs overnight inside the incubator in a humidified atmosphere of 5% CO2 at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Shear stress experiment
	<ol>
		<li>Seed 2 x 105 ECs suspended in 100 &#181;L of EC medium into the gelatin-coated flow chamber slide. Incubate the cells for 4 h at 37 &#176;C and 5% CO2 to allow the cells to attach to the culture surface.</li>
		<li>Place the perfusion set on the fluidic unit as described in the manufacturer&#39;s instructions. Fill the reservoirs and the perfusion set in&#160;about 12 mL of pre-warmed complete endothelial cell medium. Remove manually air bubbles from the perfusion set to equilibrate the level of both reservoirs at 5 mL.</li>
		<li>Place the fluidic unit with the mounted perfusion set, without the chamber slide, in the incubator and connect its electric cable to the computer-regulated pump. Remove all air bubbles from the reservoirs and perfusion set, running a predefined protocol in the pump control software. 
		NOTE. It is very important to remove air bubbles for the system to function correctly.</li>
		<li>Take the fluidic unit with the mounted perfusion set to the laminar flow hood and attach the slides with a confluent cell monolayer.</li>
		<li>Place the fluidic unit with the mounted perfusion and slides with cells in the incubator and connect its electric cable to the pump.</li>
		<li>In the pump control software, set up the following parameters: viscosity of the medium (0.0072), type of slide, perfusion set calibration factor, shear stress rate (20 dyn/cm2), type of flow (unidirectional), and time (72 h), and start the flow (see the equipment instructions for further details). Harvest the cells treated with the same media without exposure to flow as static controls.</li>
		<li>After the stimulus is completed, wash the cells once with PBS and proceed to harvest the samples according to the required assay. For fluorescent staining, see step 2.3.1, and for RNA extraction, see step 2.3.2.</li>
	</ol>
	</li>
	<li>Demonstration of the effectiveness of the shear stress protocol
	<ol>
		<li>Fluorescent staining of actin filaments (F-actin)
		<ol>
			<li>Fix and permeabilize the cells as indicated in steps 1.4.2-1.4.6. Use a volume of 100 &#181;L in the &#181;-slide.</li>
			<li>Stain the F-actin with fluorescent-labeled phalloidin (diluted at 1:200 in PBS) and counterstain the nucleus with DAPI (final concentration of 1 mg/mL in PBS) for 2 h at RT, protected from light.</li>
			<li>Wash three times with PBS.</li>
			<li>Observe the cells under a fluorescence microscope. 
			NOTE: DAPI is detected with a DAPI light cube (Ex: 335-379 nm; Em: 417-477 nm) and phalloidin is detected with a GFP light cube (Ex: 459-481 nm; Em: 500-550 nm). Light cubes with a similar range of excitation/emission wavelength are also adequate for these fluorochromes. If other fluorochromes are used, check whether the microscope used has appropriate filter sets for their detection.</li>
		</ol>
		</li>
		<li>Gene expression by real-time quantitative reverse transcription (RT-qPCR)
		<ol>
			<li>Collect the cells from the &#181;-slide with 350 &#181;L of a commercial&#160;guanidine-thiocyanate-containing lysis buffer. Isolate the total RNA with column-based extraction kits, according to the manufacturer&#39;s instructions. 
			NOTE: Due to the limited number of cells cultured in the &#181;-slide, the use of column-based extraction of RNA is recommended.</li>
			<li>Prepare cDNA using a cDNA synthesis kit with transcriptase reverse enzyme, according to the manufacturer&#39;s instructions.</li>
			<li>Verify the expression of the gene(s) regulated by shear stress: KLF2, KLF4, and NOS3. Use the housekeeper gene PPIA/cyclophilin to normalize the results. The specific primers for the reaction are listed in Table 1.</li>
			<li>Perform RT-qPCR using an SYBR green fluorescent DNA stain kit under the following reaction conditions: i) 50 &#176;C for 2 min, ii) 95 &#176;C for 15 min, iii) 94 &#176;C for 15 s, iv) 60 &#176;C for 30 s, v) 72 &#176;C for 30 s. Repeat steps iii through v for 40 cycles. Add a melting step at the end of the reaction to verify the primer&#39;s specificity.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Cyclic stretch on hSVECs
<ol>
	<li>Preparation
	<ol>
		<li>Apply silicone-based lubricant to the tops and sides of the 6-well equibiaxial loading station of 25 mm. 
		NOTE: This reduces friction between the culture plate membrane and the station, allowing better distribution of the cyclic strain. This step must be done before each experiment.</li>
	</ol>
	</li>
	<li>Cyclic stretch experiment
	<ol>
		<li>Seed 4 x 105 cells suspended in 3 mL to each well of the 6-well flexible-bottomed culture plates coated with collagen I (Table of Materials). Plan to have a plate(s) in static condition as a control group. Keep the cells in the incubator (37 &#176;C in humidified air with 5% CO2) until they reach 100% confluency (usually 48 h after seeding). Prior to the start of the stretch protocol, wash the cells once with pre-warmed PBS and add 3 mL of fresh culture medium per well.</li>
		<li>Insert the baseplate with all lubricated loading stations in the incubator and appropriately connect the tubing with the controller equipment of the tension cell stretching bioreactor system (see the equipment instructions for further details).</li>
		<li>Attach each culture plate to one red gasket, provided by the tension cell stretching bioreactor system. Then, put them into the baseplate inside the incubator.
		<ol>
			<li>Make sure the plates are fully seated, pressed, and sealed to the baseplate to avoid air leaking during the vacuum pumping. It is important to have all four culture plates in the baseplate to have the proper vacuum and the stretching loading.</li>
			<li>In case of having fewer experimental plates, use an empty one(s) to complete the four positions in the baseplate. Add the acrylic sheet (supplied with the equipment) on top of the culture plates to improve the baseplate sealing. Place the static plates in the same incubator.</li>
		</ol>
		</li>
		<li>Turn on the controller equipment and the computer system. Open the software icon and configure the desired regimen: size of the loading station to be used, type of strain, percentage of elongation, waveform shape, frequency, and time. For detailed information, see the manufacturer&#39;s instructions. Select and download the regimen. Here, the following regimen was used: 1/2 sinus waveform shape, 1 Hz frequency, 5% of elongation (to mimic venous stretch), and 15% of elongation (to mimic arterial stretch) up to 72 h.</li>
		<li>Turn on the vacuum system and click on Start on the computer screen&#160;to run the stretching. Check if the silicone membrane is moving and stretching the cells.</li>
		<li>After the stimulus is completed, wash the cells once with PBS and proceed to harvest the samples according to the required assay. Here, to demonstrate the effectiveness of cyclic stretch, collect the hSVEC culture medium, keep it at -80 &#176;C for further nitric oxide (NO) measurement, and fix the cells for fluorescent staining. 
		NOTE. The baseplate cannot be stored inside the incubator when not in use; otherwise, the temperature can modify the flat shape and make it useless.</li>
	</ol>
	</li>
	<li>Demonstration of the effectiveness of the cyclic stretch protocol
	<ol>
		<li>Fluorescent staining of F-actin
		<ol>
			<li>Fix the cells as indicated in step 1.4.3. Use a volume of 1 mL per well.</li>
			<li>Wash the cells three times with PBS. Maintain the cells in PBS so they do not dry while preparing the silicone membrane for the next steps.</li>
			<li>Use a cotton swab to remove the excess silicone-based lubricant from the bottom of the membrane. Then, gently apply window cleaner or hand soap with a new cotton swab. Repeat the process until all the lubricant is removed from the membrane. Then, clean with deionized water. 
			NOTE. It is important to completely remove the lubricant from the silicone membrane to have microscopy images of good quality.</li>
			<li>Carefully cut the silicone membranes into small pieces to fit on 8-well chamber slides for staining. Permeabilize the cells as indicated in step 1.4.5.</li>
			<li>After the washing steps, stain the F-actin using phalloidin and the nucleus with DAPI. Proceed to the analysis, as indicated in steps 2.3.1.2 and 2.3.1.3. Use a volume of 0.3 mL per chamber well.</li>
			<li>Remove the media chamber with the separator. Place one drop of mounting medium reagent (50% glycerol in PBS) and place a glass slide (24 mm x 60 mm).</li>
			<li>Observe the cells under a fluorescence microscope. 
			NOTE: DAPI is detected with a DAPI light cube (Ex: 335-379 nm; Em: 417-477 nm) and phalloidin is detected with a red fluorescent protein (RFP) light cube (Ex: 511-551 nm; Em: 573-613 nm). Light cubes with a similar range of excitation/emission wavelength are also adequate for these fluorochromes. If other fluorochromes are used, check whether the microscope used has appropriate filter sets for their detection.</li>
		</ol>
		</li>
		<li>NO measurement-estimated by the nitrite (NO2) accumulation in the hSVEC conditioned medium using a potassium iodide-based reductive chemiluminescence assay
		<ol>
			<li>Preparation
			<ol>
				<li>Prepare a standard curve from 0.01-10 mM of sodium nitrite (NaNO2 in ultrapure water) solution.</li>
				<li>Add 5 mL of acetic acid and 1 mL of potassium iodide (50 mg in 1 mL of ultrapure water) into the purge vessel of the nitric oxide analyzer.</li>
			</ol>
			</li>
			<li>NO measurement
			<ol>
				<li>Add 20 &#181;L of the NaNO2 standard curve into the purge vessel of the nitric oxide analyzer. Measure it according to the manufacturer&#39;s protocol.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Add 20 &#181;L of the conditioned medium into the purge vessel of the nitric oxide analyzer. Measure it according to the manufacturer&#39;s protocol.</li>
		<li>Calculate the NO2 concentrations based on the standard curve calibration. Adjust the values obtained by the total volume of the conditioned medium analyzed6,11.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

The saphenous vein segment should have be least 2 cm to successfully isolate hSVECs. Small segments are difficult to handle and tie the ends of the vessel to maintain the collagenase solution to isolate the cells. The reduced luminal surface area does not yield sufficient cells to expand the culture. To minimize the risk of contamination with non-ECs, the manipulation of the saphenous vein segment needs to be very gentle during the entire procedure. It is important to be careful when introducing the pipette tips into the...

Endothelial cell (EC) dysfunction is a key player in saphenous vein graft failure1,2,3,4. The sustained increase of shear stress and cyclic stretch induces the proinflammatory phenotype of human saphenous vein endothelial cells (hSVECs)3,4,5,6. The underlying molecular pathways are still not fully understood, and standardized protocols for in vitro studies may leverage the efforts for novel insights in the area. Here, we describe a simple protocol to isolate, characterize, and expand hSVECs and how to expose them to variable levels of shear stress and cyclic stretch, mimicking the venous and arterial hemodynamic conditions.
hSVECs are isolated by collagenase incubation and can be used up to passage 8. This protocol requires less manipulation of the vessel compared with the other available protocols7, which reduces contamination with smooth muscle cells and fibroblasts. On the other hand, it requires a larger vessel segment of at least 2 cm to have efficient EC extraction. In the literature, it has been reported that ECs from large vessels can also be obtained by mechanical removal7,8. Although effective, the physical approach has the disadvantages of low EC yield and higher fibroblast contamination. To increase the purity, extra steps are needed using magnetic beads or cell sorting, increasing the cost of the protocol due to the acquisition of beads and antibodies7,8. The enzymatic method has faster and better outcomes regarding EC purity and viability7,8.
The most frequently used ECs to study endothelial dysfunction are human umbilical vein endothelial cells (HUVECs). It is known that the EC phenotype changes in different vascular beds, and it is essential to develop methods that represent the vessel under investigation9,10. In this respect, the establishment of a protocol to isolate a hSVEC and culture it under mechanical stress is a valuable tool to understand the contribution of hSVEC dysfunction in vein graft disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-0.02% EDTA solution</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>15 &#181; slide I 0.4 Luer&#160;</td>
			<td>Ibidi</td>
			<td>80176</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dilactate (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>6-wells equibiaxial loading station of 25 mm&#160;</td>
			<td>Flexcell International Corporation</td>
			<td>LS-3000B25.VJW</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide with removable well</td>
			<td>Thermo Fisher Scientific</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid (Glacial)</td>
			<td>Millipore</td>
			<td>100063</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic sheet 1 cm thick</td>
			<td>Plexiglass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody</td>
			<td>Abcam</td>
			<td>ab24590</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31, FITC antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MHCD3101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-VE-cadherin antibody</td>
			<td>Cell Signaling</td>
			<td>2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioflex plates collagen I</td>
			<td>Flexcell International Corporation</td>
			<td>BF3001C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin solution</td>
			<td>Sigma-Aldrich</td>
			<td>A8412</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton suture EP 3.5 15 x 45 cm</td>
			<td>Brasuture</td>
			<td>AP524</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclophilin forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclophilin reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 basal medium</td>
			<td>Lonza</td>
			<td>CC3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 SingleQuots supplements</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>2657-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexcell FX-5000 tension system</td>
			<td>Flexcell International Corporation</td>
			<td>FX-5000T</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount aqueous mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium from porcine intestinal mucosa 5000 IU/mL</td>
			<td>Blau Farmac&#234;utica</td>
			<td>SKU 68027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi pump system (Pump + Fluidic Unit)</td>
			<td>Ibidi</td>
			<td>10902</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF2 forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF2 reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF4 forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>KLF4 reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>NOA 280 nitric oxide analyzer</td>
			<td>Sievers Instruments</td>
			<td>NOA-280i-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NOS3 forward primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>NOS3 reverse primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion set 15 cm, ID 1.6 mm, red, 10 mL reservoirs</td>
			<td>Ibidi</td>
			<td>10962</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin - Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin - Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>221945</td>
			<td></td>
		</tr>
		<tr>
			<td>QuanTitec SYBR green PCR kit</td>
			<td>Qiagen</td>
			<td>204143</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 12K flex platform&#160;</td>
			<td>Applied Biosystems</td>
			<td>4471087</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy micro kit&#160;</td>
			<td>Quiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide glass (24 mm x 60 mm)</td>
			<td>Knittel Glass</td>
			<td>VD12460Y1D.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Sigma-Aldrich</td>
			<td>31443</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV first-strand synthesis system</td>
			<td>Thermo Fisher Scientific</td>
			<td>18091200</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Type II collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
	</tbody>
</table>

human saphenous vein endothelial cell isolation and exposure to controlled levels of shear stress and stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

Typically, adhered ECs can be observed 3-4 days after extraction. hSVECs initially form clusters of cells and display a typical &#34;cobblestone&#34; morphology (Figure 1B). They express the EC markers CD31 (Figure 1C,D) and VE-cadherin (Figure 1D). hSVECs can be easily propagated on a non-coated treated cell culture dish, and they retain the endothelial phenotype in culture up to eight passages.
<p class="jove_...

Watch this Scientific Journal Video about Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch at JoVE.com

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

human-saphenous-vein-endothelial-cell-isolation-exposure-to

Research

JoVE Journal

Medicine

882 Views.  Universidade de Sao Paulo. We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Video: Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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.

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.

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

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

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 ...