Name: harvest of endothelial cells from the balloon tips of swan-ganz catheters after right heart catheterization
Uploaded: 2019-01-23T14:30:00.000+00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization at JoVE.com

The authors would like to thank the physicians and support staff of the Advanced Heart Failure and Pulmonary Hypertension service of Allegheny General Hospital for the provision of hundreds of catheter samples over several years that have facilitated the optimization of this methodology.

&#8203;All studies described in this article were approved by the Institutional Review Board of the Allegheny Health Network. All patients provided informed consent before the administration of sedative agents and were de-identified by being assigned a study number.
1. Bedside Collection
NOTE: Everything in this protocol should be done on ice, using pre-chilled solutions. It is important to induce cold stasis in the cells to preserve their in situ biological phenotype. This protocol involves the handling of blood and cells obtained from humans and therefore universal precautions should be observed.
<ol>
	<li>After withdrawal of the catheter from the sheath, immediately immerse the catheter in a 50 mL centrifuge tube containing 40 mL of chilled sterile saline. Swirl the catheter several times to remove blood, and flush the line if necessary to remove blood retained within the line.</li>
	<li>Cut off the distal 5 cm of the cath tip and allow it to fall into the 50 mL centrifuge tube containing chilled saline. Immediately remove the cath tip with forceps and transfer it to a 15 mL centrifuge tube containing 15 mL of endothelial cell (EC) growth medium.</li>
	<li>Seal the tube, place it on ice and transfer to the lab to prepare for processing or shipping. It is critical that the balloon stays immersed in medium, so make sure there is minimal headspace in the 15 mL tube by filling it completely with EC growth medium.</li>
	<li>Process the sample immediately by proceeding to Step 2.1 or seal and ship the sample on wet ice overnight for processing at a laboratory skilled in this protocol. If cells are ultimately going to be used for cell culture, it is strongly recommend that the cath tip be immediately processed at the same site at which it was collected.</li>
</ol>
2.Cell Harvest
NOTE: Everything in this protocol should be done on ice, using pre-chilled solutions. The cells are in cold stasis, and it is important to avoid awakening them until they are ready to be fixed or cultured. This entire protocol should be performed in a laminar flow hood, using sterile instruments, if the ultimate intent is culture of the harvested PAECs. If culture is not planned (e.g., flow cytometry analysis), it is not necessary to observe sterile precautions.
<ol>
	<li>Add 5 mL of cell detachment solution to a 15 mL centrifuge tube; place on ice.</li>
	<li>Draw 1 mL of EC media from that remaining in the bedside collection tube into the 1 mL syringe.</li>
	<li>Quickly complete steps 2.3 through 2.5. Grip the cut end of the catheter tip with hemostats.</li>
	<li>Carefully insert the needle of the 1 mL syringe into the &#34;white&#34; hole opposite the guide wire (&#34;black hole&#34;). Take great care to avoid injury with the needle.</li>
	<li>Transfer the cath tip into the 15 mL centrifuge tube containing the cell detachment solution.</li>
	<li>Inflate the balloon, being careful not to burst it; inflating the balloon with approximately 200 &#181;L of media works well.</li>
	<li>Allow the catheter to sit in cell detachment solution on ice for 5 min with the balloon inflated. If the balloon deflates on its own during the 5 min incubation, this is acceptable.</li>
	<li>Pour the contents of the 15 mL centrifuge tube into the 100 mm dish. Place the balloon in the cell detachment solution &#34;puddle&#34;.</li>
	<li>Scrape the inflated balloon with a cell scraper. The cell scraper can be drawn around the balloon in a circular motion or a brushing motion. Focus on the &#34;poles&#34; of the balloon, where it meets the catheter. Ensure that the cell scraper contacts the entire surface of the balloon at least twice. Rinse the cell scraper in the cell detachment solution &#34;puddle&#34; periodically.</li>
	<li>Deflate the balloon and scrape it with a brushing motion. Rinse the cell scraper in the cell detachment solution &#34;puddle&#34;.</li>
	<li>Discard the cath tip, needle and cell scraper in the appropriate waste container.</li>
	<li>Pour the cell detachment solution containing the cell harvest into a 50 mL centrifuge tube. Shake the collection tube and pour the remaining media from the collection tube into the collected sample as well.</li>
	<li>Centrifuge the sample at 650 x g at 4 &#176;C for 10 min. It is important to note that oftentimes a pellet will NOT be seen. It can be helpful to mark the location where a pellet would be seen, if visible, to avoid aspirating the pellet in the event it is too small to be seen. 
	NOTE: Normally, a pellet is only seen when the sample has been contaminated by blood. Blood contamination is immaterial to the ultimate harvest product because blood fails to bind the CD146 beads and washes through the column. Extensive testing has demonstrated that the specific brand of 1.5 mL microcentrifuge tubes referenced in the Table of Materials minimizes cell loss relative to other tubes utilized during protocol optimization.</li>
	<li>Carefully aspirate media, and avoid disturbing or aspirating the pellet.</li>
</ol>
3. Cell Purification
NOTE: Everything in this protocol should be done on ice, using pre-chilled solutions. The cells are in cold stasis, and it is important to avoid awakening them until they are ready to be fixed or cultured.
<ol>
	<li>Resuspend the pellet in 1 mL of chilled red blood cell lysis solution, and transfer it to a 1.5 mL microfuge tube; gently rock for 10 min at 4 &#176;C.</li>
	<li>Centrifuge for 5 min at 500 x g and 4 &#176;C. Aspirate the RBC lysis solution.</li>
	<li>Resuspend the pellet in 60 &#956;L of PBS, 20 &#956;L of FcR blocker, and 20 &#956;L of anti-human CD146 microbeads. Incubate for 15 min at 4 &#176;C.</li>
	<li>Add 1 mL of chilled PBS. Centrifuge for 5 min at 500 x g and 4 &#176;C. Aspirate the supernatant and resuspend the pellet in 1 mL of chilled PBS.</li>
	<li>Place a magnetic LS column on a magnetic rack (see Table of Materials for specific commercial identities of these items) at room temperature. Prime column by adding 1 mL of PBS and allow the entire volume to flow through. Discard the flow-through.</li>
	<li>Place the sample in the magnetic column. Discard the flow-through.</li>
	<li>Wash column with 3 mL of chilled PBS three times. Discard the flow-through from each wash.</li>
	<li>Add 3 mL of chilled PBS to a 15 mL centrifuge tube. Remove the magnetic column from magnet, pour the 3 mL of PBS from the 15 mL centrifuge tube into the column and quickly place the column in the centrifuge tube to catch the flow-through. Immediately plunge with the plunger supplied with column. The sample is now purified, ready for fixation/staining, or culture.</li>
</ol>

<ol>
	<li>Galie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2015+ESC/ERS+Guidelines+for+the+diagnosis+and+treatment+of+pulmonary+hypertension:+The+Joint+Task+Force+for+the+Diagnosis+and+Treatment+of+Pulmonary+Hypertension+of+the+European+Society+of+Cardiology+(ESC)+and+the+European+Respiratory+Society+(ERS):+Endorsed+by:+Association+for+European+Paediatric+and+Congenital+Cardiology+(AEPC),+International+Society+for+Heart+and+Lung+Transplantation+(ISHLT).">2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT).</a> European Respiratory Journal. 46 (4), 903-975 (2015).</li><li>Pollett, J. B., Benza, R. L., Murali, S., Shields, K. J., Passineau, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvest+of+pulmonary+artery+endothelial+cells+from+patients+undergoing+right+heart+catheterization.">Harvest of pulmonary artery endothelial cells from patients undergoing right heart catheterization.</a> Journal of Heart Lung Transplantation. 32 (7), 746-749 (2013).</li><li>Benza, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+expression+of+Bcl-2+in+pulmonary+artery+endothelial+cells+associates+with+pulmonary+arterial+hypertension+relative+to+heart+failure+with+preserved+ejection+fraction.">In situ expression of Bcl-2 in pulmonary artery endothelial cells associates with pulmonary arterial hypertension relative to heart failure with preserved ejection fraction.</a> Pulmonary Circulation. 6 (4), 551-556 (2016).</li><li>Bull, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+endothelial+cells+in+pulmonary+hypertension.">Circulating endothelial cells in pulmonary hypertension.</a> Thrombosis and Haemostasis. 90 (4), 698-703 (2003).</li><li>Kaneko, F. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+reaction+products+of+nitric+oxide+as+quantitative+markers+of+primary+pulmonary+hypertension.">Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension.</a> American Journal of Respiratory and Critical Care Medicine. 158 (3), 917-923 (1998).</li><li>Rothman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transvenous+procurement+of+pulmonary+artery+smooth+muscle+and+endothelial+cells+using+a+novel+endoarterial+biopsy+catheter+in+a+canine+model.">Transvenous procurement of pulmonary artery smooth muscle and endothelial cells using a novel endoarterial biopsy catheter in a canine model.</a> Journal of the American College of Cardiology. 27 (1), 218-224 (1996).</li><li>Savale, L., Guignabert, C., Weatherald, J., Humbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+medicine+and+personalising+therapy+in+pulmonary+hypertension:+seeing+the+light+from+the+dawn+of+a+new+era.">Precision medicine and personalising therapy in pulmonary hypertension: seeing the light from the dawn of a new era.</a> European Respiratory Reviews. 27 (148), (2018).</li><li>Kanwar, M., Raina, A., Passineau, M., Benza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+Pulmonary+Arterial+Hypertension:+Evolving+Therapeutic+Strategies.">Idiopathic Pulmonary Arterial Hypertension: Evolving Therapeutic Strategies.</a> Seminars in Respiratory and Critical Care Medicine. 38 (5), 606-618 (2017).</li><li>Elshal, M. F., Khan, S. S., Takahashi, Y., Solomon, M. A., McCoy, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD146+(Mel-CAM),+an+adhesion+marker+of+endothelial+cells,+is+a+novel+marker+of+lymphocyte+subset+activation+in+normal+peripheral+blood.">CD146 (Mel-CAM), an adhesion marker of endothelial cells, is a novel marker of lymphocyte subset activation in normal peripheral blood.</a> Blood. 106 (8), 2923-2924 (2005).</li><li>Elshal, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+population+of+effector+memory+lymphocytes+identified+by+CD146+having+a+distinct+immunophenotypic+and+genomic+profile.">A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile.</a> BMC Immunolology. 8, 29 (2007).</li><li>Newman, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PECAM-1+(CD31)+cloning+and+relation+to+adhesion+molecules+of+the+immunoglobulin+gene+superfamily.">PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.</a> Science. 247 (4947), 1219-1222 (1990).</li></ol>

MJP and RLB are inventors on patents claiming portions of the methods described.

The technique described in this article is best appreciated in the context of previous experimental paradigms for studying the pulmonary vascular endothelium. Previous attempts to obtain PAECs from humans have been limited to explanted/cadaveric lungs5 and a short-lived attempt to develop a catheter capable of pulmonary artery biopsy6. As noted in a recent review7, the present technique is distinguished from lung tissue harvest by its ability to obtain PAECs from a living human, and to do so at multiple time points in the progression of the disease and/or therapy. However, it should be noted that a companion technique has recently been reported whereby induced pluripotent stem cells (IPSCs) can be transdifferentiated into PAECs4,5. This technique produces abundant cultures of patient-specific PAECs and allows study of the effects of patient-specific genetic background on PAEC biology. Despite these advantages, these IPSC-derived PAECs cannot offer any information about the interaction between local microenvironment and patient-specific genetic background. Our own group has presented the view that the "cath tip" and IPSC techniques are likely to be synergistic in advancing the field8.
Within this present and historical context, the limitations of this technique should be noted. First, it is important to note that yields of PAECs derived from this method can vary considerably. Presumably, patient-, disease state- and physician-specific factors contribute to this variability, as the protocol does not contain steps that would be expected to result in major loss of source material. Table 1 shows representative CD146+ yields from a variety of PAEC harvests utilizing catheter tips obtained from a variety of patients with different disease states of pulmonary hypertension and a variety of physician operators. Second, cell yields are overall low, and their viability for culture has proven to be lower yet in our hands. While we have previously grown cultures of PAECs using this method1, we assume those results represent rescue of a subset of progenitor cells following die-off of most of the sample and is only intermittently successful in our hands. For example, it is not unusual to find only a few dozen adherent PAECs in culture the morning after a harvest has been plated. Often these micro-cultures do not survive to expansion. Further work is needed to optimize this protocol in a manner that will sustain the viability of PAECs and their conditions in culture to achieve the desired aim of propagation of patient-specific PAEC harvests. Finally, the CD146 positive selection we have adapted cannot exclude contamination with other CD146+ cells, such as circulating endothelial cells4, pericytes, smooth muscle cells and rare CD146+ T cells9,10. Nevertheless, these cells are presumed to be very few in number in samples collected in this manner, and CD146 is considerably more specific for this protocol than other cell markers of endothelial cells such as CD31 (which is also present on a variety of blood-borne cells including platelets11). Any future improvements aimed at improving specificity should be balanced against their potentially negative effect on yield.

Current U.S. and European guidelines require right heart catheterization for the diagnosis of pulmonary hypertension1. Physicians and scientists seeking to understand this disease desire biological samples from the pulmonary vasculature to complement the hemodynamic data that is gathered during RHC. Unfortunately, the risk associated with pulmonary vascular biopsy is very high, and therefore biological samples of the pulmonary vasculature are rarely if ever obtained from living patients undergoing RHC. While much insight has been gained from pulmonary vascular tissue obtained from cadaveric or explanted lungs, these samples are unable to reflect the biology of the PH disease process as it is unfolding in a living patient. In 2013, we first reported that pulmonary artery endothelial cells (PAECs) are denuded from the branches of the pulmonary artery by physical contact with the balloon tip of the Swan-Ganz catheter during RHC, and can be recovered in small numbers from the catheter tips after the procedure2.
This article illustrates our technique for the removal and purification of pulmonary artery endothelial cells (PAECs) from the balloon tips of Swan-Ganz catheters following right heart catheterization. Heretofore, the methodology for performing this harvest technique has been described in insufficient detail in the literature to ensure uniform results between laboratories performing the technique. This article is intended to overcome this deficiency, offering visualized demonstration of the technique, from bedside collection of catheter tips to the final product of purified CD146+ endothelial cells. This technique should aid in advancing research into the pathobiology of various etiologies of PH, and possibly result in the development of new diagnostic assays to complement hemodynamic data gathered during RHC.
The importance of this technique to the field of pulmonary vascular medicine and research is that it provides access to a snapshot of the biology of the pulmonary endothelium in situ and in contrast to earlier studies on explanted or cadaveric lungs, does not represent an endpoint for the subject&#39;s lung. Recently, we have shown that an anti-apoptotic biomarker in these cells may have usefulness in more precisely phenotyping related varieties of pulmonary hypertension3. The yield of cells obtained is relatively small, ~5,000-25,000 PAECs per sample, and these cells are difficult (but not impossible) to culture. We believe that clever clinicians and researchers will find synergies between these companion techniques in order to advance pulmonary vascular research, diagnostics and prognostics.

<table><tbody><tr><td>100 mm culture dish</td><td>Fisher</td><td>NC9385690</td><td></td></tr><tr><td>15 mL Centrifuge Tubes</td><td>Midsci</td><td>C15R</td><td></td></tr><tr><td>16% Formaldehyde</td><td>Fisher</td><td>PI28908</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Midsci</td><td>C50R</td><td></td></tr><tr><td>Accutase</td><td>Thermo</td><td>A1110501</td><td>cell detachment solution</td></tr><tr><td>Cell Scraper</td><td>Fisher</td><td>08-100-241</td><td></td></tr><tr><td>Endothelial Cell Growth media</td><td>Cell Applications</td><td>211-500</td><td></td></tr><tr><td>Forceps</td><td>any</td><td></td><td></td></tr><tr><td>Hemostat</td><td>any</td><td></td><td></td></tr><tr><td>LoBind microfuge tube</td><td>Fisher</td><td>13-698-794&amp;nbsp;</td><td></td></tr><tr><td>Miltenyi CD146 beads</td><td>Miltenyi</td><td>130-093-596</td><td></td></tr><tr><td>Miltenyi FCR blocker</td><td>Miltenyi</td><td>130-059-901</td><td></td></tr><tr><td>Miltenyi LS columns</td><td>Miltenyi</td><td>130-042-401</td><td></td></tr><tr><td>Miltenyi magnet</td><td>Miltenyi</td><td>MidiMACS</td><td></td></tr><tr><td>Miltenyi RBC lysis buffer</td><td>Miltenyi</td><td>130-094-183</td><td></td></tr><tr><td>PBS</td><td>Fisher</td><td>BP2438-4</td><td></td></tr><tr><td>Scissors</td><td>any</td><td></td><td></td></tr><tr><td>Syringes</td><td>BD</td><td>309597</td><td></td></tr></tbody></table>

harvest of endothelial cells from the balloon tips of swan-ganz catheters after right heart catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol results in the purification of cells, from Swan-Ganz catheter tips after right heart catheterization, that bind to a CD146 selection column and have forward/side scatter profiles that are indistinguishable from cultured primary human pulmonary artery endothelial cells (Figure 1). Given that the catheter balloon is in physical contact with only the introducer sheath (a cell-free material), the pulmonary artery vessel wall, and the blood, data is provided herein to demonstrate that these cells are not derived from the blood (Figure 2), and therefore it is assumed that they are indeed derived from the pulmonary artery vessel wall. The reader should note that the anti-CD146 magnetic beads that are used to purify the cells remain adherent to the cell surface after the procedure, and thus it is suboptimal to attempt to analyze the cell harvests for CD146 as a marker of endothelial cells. CD31 or other endothelial markers would presumably be available for labeling and could be considered as alternatives.
Key elements of this protocol have been identified that may affect results. First, it is important to rinse the catheter tip at the bedside in order to remove blood. Figure 3 shows Side scatter (SSC)/Forward scatter (FSC) profiles of flow-through from the anti-CD146 column for one sample that was properly rinsed and one that was not. The column appears to exclude blood-derived cells from the harvests (Figure 2), but the rinsing step will ensure minimal possible contamination from circulating endothelial cells (which are normally present at &#60;50 cells/mL of blood4). We have observed that eluate from the CD146 column is completely clear, even in the case of very bloody catheter tip samples. This suggests that the column is able to handle significant amounts of blood without clogging or retaining non-CD146+ bloodborne cells.
Second, it is important to inflate the balloon tip at least once during the cell detachment solution/cell scraper step. Given that the putative PAECs on the balloon tip remain adherent despite the saline rinse step, it seems likely they may be partially trapped by folds in the vinyl balloon as it is deflated in preparation for removal from the patient. Presumably, the inflation step frees the PAECs, allowing them to be gently scraped away. It is not immediately clear one might test this hypothesis, but lower yields of cells have been observed when the balloon is not inflated (data not shown).
Finally, it is important to note that yields of PAECs derived from this method can vary considerably. Presumably, patient-, disease state- and physician-specific factors contribute to this variability, as the protocol does not contain steps that would be expected to result in major loss of source material. Table 1 shows representative CD146+ yields from a variety of PAEC harvests utilizing catheter tips obtained from a variety of patients with different disease states of pulmonary hypertension and a variety of physician operators.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58353/58353fig1.jpg" /> 
Figure 1: SSC/FSC profiles of a typical catheter balloon tip harvest from a pulmonary hypertension patient (A) and a similar number of cells from a commercially-acquired primary human PAEC culture (B). Both samples were purified with the anti-CD146 column, fixed, and permeablized as described in the protocol, prior to FACS analysis. <a href="https://www.jove.com/files/ftp_upload/58353/58353fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58353/58353fig2.jpg" /> 
Figure 2: SSC/FSC profiles of a typical catheter balloon tip harvest from a pulmonary hypertension patient (A) and serial 1:10 dilution of Ficoll gradient-purified human buffy coat (B, C, D). (A) was purified with the anti-CD146 column whereas (B-D) were not. All cell samples were fixed and permeablized as described in the protocol, prior to FACS analysis. <a href="https://www.jove.com/files/ftp_upload/58353/58353fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58353/58353fig3.jpg" /> 
Figure 3: SSC/FSC profiles of a catheter balloon tip harvest from a pulmonary hypertension patient in which blood was visible on the catheter tip prior to processing. Prior to step 3.1 of the protocol, the sample was split and lysis buffer was added to one aliquot and withheld from the other. (A) and (C) show the SSC/FSC profiles of the lysed sample, both the cells retained by the column (A) and the flow-through from the column. (B) and (D) show the SSC/FSC profiles of the unlysed sample, both the cells retained by the column (B) and the flow-through from the column (D). We conclude that lysis removes some but not all of the leukocytes but that leukocytes are not retained by the column and are thus excluded from the final harvest, regardless of lysis. <a href="https://www.jove.com/files/ftp_upload/58353/58353fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Date</td>
			<td>Cell Yield</td>
			<td>Diagnosis</td>
		</tr>
		<tr>
			<td>433</td>
			<td>4/17/2018</td>
			<td>7364</td>
			<td>PH-Interstitial lung disease</td>
		</tr>
		<tr>
			<td>279</td>
			<td>4/17/2018</td>
			<td>7130</td>
			<td>PH-Scleroderma</td>
		</tr>
		<tr>
			<td>432</td>
			<td>4/13/2018</td>
			<td>3976</td>
			<td>PH-Multifactorial</td>
		</tr>
		<tr>
			<td>431</td>
			<td>4/13/2018</td>
			<td>3634</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>390</td>
			<td>4/13/2018</td>
			<td>5666</td>
			<td>PAH</td>
		</tr>
		<tr>
			<td>423</td>
			<td>3/23/2018</td>
			<td>9024</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>272</td>
			<td>3/23/2018</td>
			<td>7098</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>408</td>
			<td>2/27/2018</td>
			<td>4784</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>403</td>
			<td>2/14/2018</td>
			<td>10376</td>
			<td>PH-ASD+mitral disease</td>
		</tr>
		<tr>
			<td>396</td>
			<td>1/26/2018</td>
			<td>4890</td>
			<td>HFpEF-IPCPH</td>
		</tr>
		<tr>
			<td>397</td>
			<td>1/26/2018</td>
			<td>7672</td>
			<td>Normal hemodynamics</td>
		</tr>
		<tr>
			<td>318</td>
			<td>1/4/2018</td>
			<td>7002</td>
			<td>HFrEF-LVAD present</td>
		</tr>
	</tbody>
</table>
Table 1: CD146+ yields from a variety of PAEC harvests. Representative CD146+ yields from a variety of PAEC harvests utilizing catheter tips obtained from a variety of patients with different disease states of pulmonary hypertension and a variety of physician operators.

Watch this Scientific Journal Video about Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization at JoVE.com

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

harvest-endothelial-cells-from-balloon-tips-swan-ganz-catheters-after

Research

JoVE Journal

Medicine

8.2K Views.  Allegheny Health Network. This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body. 

Video: Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

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 ...

Biology

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Bioengineering

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Immunology and Infection

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

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

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.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Developmental Biology

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.

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 ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

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 ...

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although ...