This research is supported by the NSF CAREER award, the Hartwell Foundation, and the American Heart Association (#0830384N).

1. Preparation
<ol>
	<li>Autoclave in a covered instrument tray the following items: 
	<ol>
		<li>Serrated tissue forceps - For handling the leaflet tissue</li>
		<li>Tissue scissors (8 cm) - For trimming leaflet tissue and cusps</li>
		<li>Cotton Swabs - For isolating the endothelial layer from the leaflet or cusp</li>
	</ol>
	</li>
	<li>Make sterile collagenase solution 
	<ol>
		<li>Add 4.0 grams of powdered DMEM to 250 mL of 18 M&Omega; water.</li>
		<li>Add 1.11 grams of sodium bicarbonate.</li>
		<li>Add (600 U/mL) 180,000 units of collagenase.</li>
		<li>Add 1% (3mL) Penicillin/Streptomycin.</li>
		<li>Adjust the pH of the solution to 7.2.</li>
		<li>Bring the solution up to 270 mL.</li>
		<li>Sterilize the solution by passing through a 0.2 &mu;m filter.</li>
		<li>Add 10% (30mL) of sterile fetal bovine serum.</li>
 </ol>
 </li>
	<li>Make sterile endothelial cell medium 
	<ol>
		<li>Add 6.7 grams of powdered DMEM to 400 mL of 18 M&Omega; water.</li>
		<li>Add 1.85 grams of sodium bicarbonate.</li>
		<li>Add (50 U/mL) 25,000 units of heparin.</li>
		<li>Add 1% (5mL) Penicillin/Streptomycin.</li>
		<li>Adjust the pH of the solution to 7.2.</li>
		<li>Bring the solution up to 450 mL.</li>
		<li>Sterilize the solution by passing through a 0.2 &mu;m filter.</li>
		<li>Add 10% (50mL) of sterile fetal bovine serum.</li>
	</ol>
 </li>
	<li>Make sterile interstitial cell medium 
	<ol>
		<li>Add 13.37 grams of powdered DMEM to 800 mL of 18 M&Omega; water.</li>
		<li>Add 3.7 grams of sodium bicarbonate.</li>
		<li>Add 1% (10mL) Penicillin/Streptomycin.</li>
		<li>Adjust the pH of the solution to 7.2.</li>
		<li>Bring the solution up to 900 mL.</li>
		<li>Sterilize the solution by passing through a 0.2 &mu;m filter.</li>
		<li>Add 10% (100mL) of sterile bovine serum.</li>
	</ol>
 </li>
</ol>
2. Isolation of Heart Valve Leaflets
<ol>
	<li>Excise the aortic root immediately and aseptically from the heart after sacrifice.</li>
	<li>Thoroughly rinse the aortic root of blood with cold sterile DPBS. It is imperative to remove all blood components as soon as possible to limit VEC death and bacterial contamination. Antibiotics and antimycotics are not advised at this stage since they are potentially harmful to the VEC.</li>
	<li>Isolate valve leaflets (3 per valve) directly from the root and place in a sterile 15 mL conical tube filled with 12 mL cold DPBS. Shake several times to remove debris and refill with fresh DPBS.</li>
	<li>Transport back to the lab on ice.</li>
	<li>Upon arrival at the laboratory, place the container with the tissue under the sterile hood.</li>
</ol>
3. Isolation of Endothelial Layer
<ol>
	<li>Fill a sterile 35mm dish with 3mL of cold collagenase solution per valve (3 leaflets).</li>
	<li>Place all three leaflets from the 15 mL tube into the dish filled with the collagenase solution.</li>
	<li>Incubate the tissue for 5-10 minutes at 37&deg;C.</li>
	<li>Gently remove the endothelial layer by rotating a dry sterile swab onto the surface of the leaflet. The direction of rotation and amount of shear applied is critical for the purity of your sample. The rotation of the swab should be in an opposite direction to linear motion of your hand creating a controlled shear. This shear is what lifts the endothelial cells from the tissue. The amount of force applied should be enough to feel the resistance of the tissue but, not penetrate the basement membrane.</li>
	<li>Occasionally, dab the swab within the collagenase solution to dislodge cells from the tip fibers. After swabbing is complete, the texture of the endothelial layer should feel slightly smoother than before.</li>
	<li>Collect the cell suspension/collagenase and transfer to a new sterile 15mL tube.</li>
	<li>Centrifuge the tubes at 1000 rpm for 5 minutes to pellet any isolated cells and aspirate supernatant. If isolating interstitial cells as well, perform that protocol while these cells are being centrifuged.</li>
	<li>Add 3 mL of endothelial porcine medium to the 15 mL tubes, centrifuge a second time, and aspirate media. This second centrifugation helps filter some of the unwanted material such as tip fibers.</li>
	<li>Re-suspend the centrifuged endothelial cells in 5 mL of endothelial porcine medium and plate the cells in a pre-coated T-25 flask with collagen (use 1 flask per centrifuge tube).</li>
	<li>Let the cells grow at least 2-3 days before changing the endothelial medium. This helps the cells recover and divide since the isolation process is fairly harsh and the cell yield may be low. It is critical to passage cells near confluence since contact inhibition could lead to cell transformation.</li>
</ol>
4. Preparation of 60 mm Culture Dish for Side Specific Isolation
<ol>
	<li>Line the 60 mm glass petri dishes with aluminum foil (2 glass dishes per leaflet). The aluminum foil helps to remove the paraffin, so that the glass petri dish can be reused for other isolations.</li>
	<li>Place paraffin beads within the dishes, about half full, and cover for autoclaving.</li>
	<li>Once the autoclaving is complete and paraffin is melted, move the dish ensemble to a cool flat surface. As the paraffin cools, it will harden and create a layer that will support needle punctures.</li>
	<li>After 30 minutes, the sterilized dish can then be used as an isolation chamber to immobilize the leaflet tissue.</li>
</ol>
5. Isolation of Side Specific Endothelial Layer
<ol>
	<li>Remove the leaflets from the 15mL tubes and place on prepared 60mm culture dish for side specific isolation.</li>
	<li>Manipulate the leaflet so that the ventricularis side is face down on the paraffin surface leaving the fibrosa side exposed. Pin the edges of the leaflet to expose the endothelial layer.</li>
	<li>Place a few drops of cold collagenase on each (upwards-facing) endothelial surface and incubate the tissue for 5-10 minutes at 37&deg;C.</li>
	<li>As before, gently remove the endothelial layer by rotating a dry sterile swab onto the surface of the leaflet. The direction of rotation and amount of shear applied is critical for the purity of your sample. The rotation of the swab should be in an opposite direction to linear motion of your hand creating a controlled shear. This shear is what lifts the endothelial cells from the tissue and nothing else. The amount of force applied should be enough to feel the resistance of the tissue but, does not penetrate within the tissue.</li>
	<li>Occasionally, dab the swab within the collagenase solution to dislodge cells from the tip fibers. After swabbing is complete, the texture of the endothelial layer should feel slightly smoother than before.</li>
	<li>Collect the cell suspension/collagenase and transfer to a new sterile 15mL tube. Indicate side specificity on label (leaflets from the same valve can be pooled together).</li>
	<li>Transfer the leaflets to a new culture dish so that the ventricularis side is now exposed and repeat steps (5.3-5.6).</li>
	<li>Once all cell suspension/collagenase is collected, centrifuge the tubes at 1000 rpm for 5minutes to pellet any isolated cells and aspirate supernatant. If isolating interstitial cells as well, perform that protocol while these cells are being centrifuged.</li>
	<li>Add 3 mL of endothelial porcine medium to the tubes, centrifuge a second time, and aspirate media. This second centrifugation helps filter some of the unwanted material such as tip fibers.</li>
	<li>Re-suspend the centrifuged endothelial cells in 5 mL of endothelial porcine medium and plate the cells in a pre-coated T-25 flask with collage (use 1 flask per centrifuge tube).</li>
	<li>Let the cells grow at least 2-3 days before changing the endothelial medium. This helps the cells recover and divide since the isolation process is fairly harsh. If you notice the yield to be very low, consider moving to a smaller flask or well plate since cell-cell adhesion promotes cell growth. Remember to passage near confluence since contact inhibition could lead to cell transformation.</li>
</ol>
6. Isolation of Interstitial Cells
<ol>
	<li>Fill a sterile 15 mL centrifuge tube with 10 mL of collagenase solution per valve (3 leaflets).</li>
	<li>After swabbing the leaflets of endothelial cells, immediately place them in the appropriate 15mL tube with the collagenase solution.</li>
	<li>Incubate for approximately 12 to 18 hours (agitate gently if desired).</li>
	<li>Mix the degraded tissue gently with a serological pipette until cell suspension/collagenase becomes homogenized. This homogenization helps break up the tissue and release the interstitial cells.</li>
	<li>Centrifuge the digested tissue for 5 minutes at 1000 rpm and aspirate the supernatant.</li>
	<li>Add 5 mL interstitial porcine medium to the 15mL tubes, centrifuge a second time, and aspirate supernate.</li>
	<li>Re-suspend the centrifuged endothelial cells in 5 mL of interstitial porcine medium and plate the cells in a T-75 flask (use 1 flask per centrifuge tube).</li>
	<li>Let the cells grow at least 1-2 days before changing the interstitial cell medium. There will be much more tissue debris than with the endothelial cells but, that is expected. The cells should also grow to confluence faster than the endothelial cells because of the cell yield and their nature.</li>
</ol>
7. Representative Images
<img src="/files/ftp_upload/2158/2158fig1.jpg" alt="Figure 1" /> Figure 1. The morphology of isolated cells at 2-3 days post isolation. (A) VEC exhibit a typical endothelial morphology and form clusters to promote growth . (B) VIC morphology is similar to myofibroblasts which are generally spindle shaped and spread evenly throughout the flask.
<img src="/files/ftp_upload/2158/2158fig2.jpg" alt="Figure 2" /> Figure 2. The morphology of isolated cells at confluency. (A) VEC exhibit a typical endothelial morphology which are generally cobblestone and growth contact inhibited. (B) VIC morphology is similar to myofibroblasts which are generally spindle shaped and not growth contact inhibited.
<img src="/files/ftp_upload/2158/2158fig3.jpg" alt="Figure 3" /> Figure 3. The function characteristics of isolated cells6. (A) VEC are associated with high levels of acetylated LDL uptake (red). (B) VIC are associated with low levels of acetylated LDL uptake.
<img src="/files/ftp_upload/2158/2158fig4.jpg" alt="Figure 4" /> Figure 4. Cellular markers of isolated cells 6. (A) VEC phenotype is contributed to positive staining for Von Willebrand Factor (green), blue (nuclei). (B) VIC phenotype is contributed to negative staining for Von Willebrand Factor.
<img src="/files/ftp_upload/2158/2158fig5.jpg" alt="Figure 5" /> Figure 5. Cellular markers of isolated cells. (A) VEC phenotype is contributed to negative staining for alpha SMA (green), blue (nuclei). (B) VIC phenotype is contributed to positive staining for alpha SMA.
<table border="1">
 <tbody>
 <tr>
 <td>Dissociation Agent</td>
 <td>Dissociation Technique</td>
 <td>Cell Collection</td>
 <td>Cell Quantity</td>
 <td>Cell 
 Purity</td>
 <td>Contamination</td>
 </tr>
 <tr>
 <td>EDTA (3mM) without CaCl2</td>
 <td>5, 20, 60 min. 
 before CaCl2 addition</td>
 <td>20, 60, 120 min. before collection</td>
 <td>-</td>
 <td>+/-</td>
 <td>+++</td>
 </tr>
 <tr>
 <td>EDTA (6mM) without CaCl2</td>
 <td>5, 20, 60 min. 
 before CaCl2 addition</td>
 <td>20, 60, 120 min. before collection</td>
 <td>+/-</td>
 <td>+</td>
 <td>+++</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA (0.5 g/L)</td>
 <td>5, 10, 15 min. 
 before deactivation</td>
 <td>Medium collected immediately </td>
 <td>+</td>
 <td>+</td>
 <td>++</td>
 </tr>
 <tr>
 <td>Collagenase II (300 U/ mL)</td>
 <td>5, 10, 15 min. 
 before deactivation</td>
 <td>Medium collected immediately</td>
 <td>-, +, ++</td>
 <td>-, ++, +</td>
 <td>+</td>
 </tr>
 <tr>
 <td>Collagenase II (600 U/ mL)</td>
 <td>5, 10, 15 min. 
 before deactivation</td>
 <td>Medium collected immediately </td>
 <td>+, ++, + 
 ++</td>
 <td>++, ++, 
 -</td>
 <td>-</td>
 </tr>
 </tbody>
</table>
Table 1. Results of preliminary valvular endothelial cell isolation protocols.

<ol>
	<li>Thompson, R. P., Fitzharris, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+truncus+arteriosus+of+the+chick+embryo+heart:+the+formation+and+migration+of+mesenchymal+tissue.">Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.</a> Am J Anat. 154, 545-556 (1979).</li><li>Johnson, C. M., Fass, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+cardiac+valvular+endothelial+cells+in+culture:+A+relative+deficiency+of+fibronectin+synthesis+in+vitro.">Porcine cardiac valvular endothelial cells in culture: A relative deficiency of fibronectin synthesis in vitro.</a> Lab Invest. 49 (5), 589-598 (1983).</li><li>Manduteanu, I., Popov, D., Radu, A., Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calf+cardiac+valvular+endothelial+cells+in+culture:+production+of+glycosaminoglycans,+prostacyclin+and+fibronectin.">Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.</a> J Mol Cell Cardiol. 20 (2), 103-118 (1988).</li><li>Cheunyg, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+isolating+and+purifying+porcine+aortic+valve+endothelial+cells.">Techniques for isolating and purifying porcine aortic valve endothelial cells.</a> JHVD. 17 (6), 674-681 (2008).</li><li>Paranya, G., Vineberg, S., Dvorin, E., Kaushal, S., Roth, S. J., Rabkin, E., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> Am J Pathol. 159 (4), 1335-1343 (2001).</li><li>Butcher, J. T., Penrod, A., Garcia, A. J. M., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+morphology+and+focal+adhesion+development+of+valvular+endothelial+cells+in+static+and+fluid+flow+environments.">Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments.</a> Arterioscler Thromb Vasc Bio. 24 (1), 1429-1434 (2004).</li><li>Simmons, C. A., Grant, G. R., Manduchi, E., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+heterogeneity+of+endothelial+phenotypes+correlates+with+side-specific+vulnerability+to+calcification+in+normal+porcine+aortic+valves.">Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.</a> Circ Res. 96, 792-799 (2005).</li><li>Butcher, J. T., Tressel, S., Johnson, T., Turner, D., Sorescu, G., Jo, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Profiles+of+Valvular+and+Vascular+Endothelial+Cells+Reveal+Phenotypic+Differences:+Influence+of+Shear+Stress.">Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress.</a> Arterioscler Thromb Vasc Biol. 26, 69-69 (2006).</li><li>Parachuri, S., Yang, J. H., Aikawa, E., Melero-Martin, J. M., Khan, Z. A., Loukogeorgakis, S., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Pulmonary+Valve+Progenitor+Cells+Exhibit+Endothelial/Mesenchymal+Plasticity+in+Response+to+Vascular+Endothelial+Growth+Factor-A+and+Transforming+Growth+Factor-&#946;2.">Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-&#946;2.</a> Circ Res. 99 (8), 861-869 (2006).</li><li>Shi, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+circulating+bone+marrow-derived+endothelial+cells.">Evidence for circulating bone marrow-derived endothelial cells.</a> Blood. 92, 362-367 (1998).</li><li>Rehman, J., Li, J., Orschell, C. M., March, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+endothelial+progenitor+cells+are+derived+from+monocyte/macrophages+and+secrete+angiogenic+growth+factors.">Peripheral blood endothelial progenitor cells are derived from monocyte/macrophages and secrete angiogenic growth factors.</a> Circulation. 107, 1164-1169 (2003).</li></ol>

No conflicts of interest declared.

An understanding of valvular biology has been impaired by technical difficulties isolating and culturing pure populations of valvular endothelial cells. Typical isolation techniques involve enzymatic digestion of the underlying basal matrix or chemical dissociation of endothelial adhesive bonds2,3. Preliminary isolation experiments were qualitatively assessed by varying dissociation agents and incubation periods. The results of these experiments showed that EDTA (or Trypsin-EDTA) incubation for up to 60 min...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco&#x2019;s Modified Eagle Medium</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>50-103-PB</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H4784-1G</td>
<td>&nbsp;</td>
<tr>
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<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
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<td>Gibco</td>
<td>21300-058</td>
<td>&nbsp;</td>
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<td>Rat Tail Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>VWR</td>
<td>89031-270</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>55761</td>
<td>&nbsp;</td>
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<td>T25 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353018</td>
<td>&nbsp;</td>
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<td>T75 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353136</td>
<td>&nbsp;</td>
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<td>Precision Glide Needles</td>
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<td>305165</td>
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<td>500 mL Nalgene Filters</td>
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<td>VWR</td>
<td>73520-985</td>
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<td>1L Nalgene Filters</td>
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<td>73520-986</td>
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<td>FSC Tweezers #5</td>
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<td>Fine Science Tools</td>
<td>11295-00</td>
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isolation of valvular endothelial cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

Watch this Scientific Journal Video about Isolation of Valvular Endothelial Cells at JoVE.com

Isolation of Valvular Endothelial Cells

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

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JoVE Journal

Biology

20.2K Views.  Cornell University. We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward. 

Video: Isolation of Valvular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...