Name: Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch (Video) | JoVE Journal
Uploaded: 2023-04-21T14:35:00.000+00:00
Duration: 9 min 10 s
Description: Learn to isolate human saphenous vein endothelial cells using collagenase digestion and culture them under controlled shear stress and cyclic stretch conditions. This method enables study of mechanical stress responses in vascular biology.

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5 minutes

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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<li>Carneiro, A. P., Fonseca-Alaniz, M. H., Dallan, L. A. O., Miyakawa, A. A., Krieger, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B2-arrestin+is+critical+for+early+shear+stress-induced+Akt%2FeNOS+activation+in+human+vascular+endothelial+cells%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">β-arrestin is critical for early shear stress-induced Akt/eNOS activation in human vascular endothelial cells.</a> Biochemical and Biophysical Research Communications. 483 (1), 75-81 (2017).</li>
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<li>Beamish, J. A., He, P., Kottke-Marchant, K., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+regulation+of+contractile+smooth+muscle+cell+phenotype%3A+implications+for+vascular+tissue+engineering%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+B%2C+Reviews%22%5BJournal%5D)">Molecular regulation of contractile smooth muscle cell phenotype: implications for vascular tissue engineering.</a> Tissue Engineering. Part B, Reviews. 16 (5), 467-491 (2010).</li>
</ol>

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><tbody><tr><td>0.25% Trypsin-0.02% EDTA solution</td><td>Gibco</td><td>25200072</td><td></td></tr><tr><td>15 &amp;micro; slide I 0.4 Luer&amp;nbsp;</td><td>Ibidi</td><td>80176</td><td></td></tr><tr><td>4&#039;,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&amp;nbsp;</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&amp;ecirc;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&amp;nbsp;</td><td>Applied Biosystems</td><td>4471087</td><td></td></tr><tr><td>RNeasy micro kit&amp;nbsp;</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

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 "cobblestone" 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.
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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 ...

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Research

JoVE Journal

Medicine

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

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

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

On-Chip Endothelial Inflammatory Phenotyping

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.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

Learn to isolate human saphenous vein endothelial cells using collagenase digestion and culture them under controlled shear stress and cyclic stretch conditions. This method enables study of mechanical stress responses in vascular biology.

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

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