This work was supported by funds from the Italian Ministry of the University and Research (ex 60% 2021 and 2022) to R. P. and by grants from the Italian Cystic Fibrosis Foundation (&#65279;FFC#23/2014) and from the Italian Ministry of Health (&#65279;L548/93) to M. R.

This study was approved, and the protocol followed the guidelines of the human research ethics committee of the University of Chieti-Pescara (#237_2018bis). Figure 1 illustrates the isolation of endothelial cells from segments (1-3 cm long) of pulmonary parenchyma or pulmonary artery from deidentified human subjects (with written consent) undergoing thoracic surgery for various reasons, such as pneumothorax or lobectomy. In this latter case, the surgeons also collected a pulmonary artery seg...

<ol>
	<li>Muller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial-cell+interactions+in+leukocyte+transmigration+and+the+inflammatory+response.">Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response.</a> Trends in Immunology. 24 (6), 326-333 (2003).</li><li>Sumpio, B. E., Timothy Riley, J., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+focus:+Endothelial+cell.">Cells in focus: Endothelial cell.</a> The International Journal of Biochemistry &amp; Cell Biology. 34 (12), 1508-1512 (2002).</li><li>L&#252;scher, T. F., Tanner, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+regulation+of+vascular+tone+and+growth.">Endothelial regulation of vascular tone and growth.</a> American Journal of Hypertension. 6, 283-293 (1993).</li><li>Marki, A., Esko, J. D., Pries, A. R., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+endothelial+surface+layer+in+neutrophil+recruitment.">Role of the endothelial surface layer in neutrophil recruitment.</a> Journal of Leukocyte Biology. 98 (4), 503-515 (2015).</li><li>Crampton, S. P., Davis, J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+umbilical+vein+endothelial+cells+(HUVEC).">Isolation of human umbilical vein endothelial cells (HUVEC).</a> Journal of Visualized Experiments. (3), e183 (2007).</li><li>Ganguly, A., Zhang, H., Sharma, R., Parsons, S., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+umbilical+vein+endothelial+cells+and+their+use+in+the+study+of+neutrophil+transmigration+under+flow+conditions.">Isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions.</a> Journal of Visualized Experiments. (66), e4032 (2012).</li><li>Dejana, E., Corada, M., Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-to-cell+junctions.">Endothelial cell-to-cell junctions.</a> FASEB Journal. 9 (10), 910-918 (1995).</li><li>Plebani, R., 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=Establishment+and+long-term+culture+of+human+cystic+fibrosis+endothelial+cells.">Establishment and long-term culture of human cystic fibrosis endothelial cells.</a> Laboratory Investigation. 97 (11), 1375-1384 (2017).</li><li>Totani, 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=Mechanisms+of+endothelial+cell+dysfunction+in+cystic+fibrosis.">Mechanisms of endothelial cell dysfunction in cystic fibrosis.</a> Biochimica Et Biophysica Acta. Molecular Basis of Disease. 1863 (12), 3243-3253 (2017).</li><li>Gaskill, C., Majka, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-yield+isolation+and+enrichment+strategy+for+human+lung+microvascular+endothelial+cells.">A high-yield isolation and enrichment strategy for human lung microvascular endothelial cells.</a> Pulmonary Circulation. 7 (1), 108-116 (2017).</li><li>Hewett, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+human+endothelial+cells+from+micro-+and+macro-vessels.">Isolation and culture of human endothelial cells from micro- and macro-vessels.</a> Methods in Molecular Biology. 1430, 61 (2016).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nature Protocols. 3 (6), 1085-1091 (2008).</li><li>Comhair, S. A. 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=Human+primary+lung+endothelial+cells+in+culture.">Human primary lung endothelial cells in culture.</a> American Journal of Respiratory Cell and Molecular Biology. 46 (6), 723-730 (2012).</li><li>Visner, G. 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=Isolation+and+maintenance+of+human+pulmonary+artery+endothelial+cells+in+culture+isolated+from+transplant+donors.">Isolation and maintenance of human pulmonary artery endothelial cells in culture isolated from transplant donors.</a> The American Journal of Physiology. 267, 406-413 (1994).</li><li>Mackay, L. S., 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=Isolation+and+characterisation+of+human+pulmonary+microvascular+endothelial+cells+from+patients+with+severe+emphysema.">Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema.</a> Respiratory Research. 14 (1), 23 (2013).</li><li>Ventetuolo, C. E., 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=Culture+of+pulmonary+artery+endothelial+cells+from+pulmonary+artery+catheter+balloon+tips:+considerations+for+use+in+pulmonary+vascular+disease.">Culture of pulmonary artery endothelial cells from pulmonary artery catheter balloon tips: considerations for use in pulmonary vascular disease.</a> The European Respiratory Journal. 55 (3), 1901313 (2020).</li><li>Wang, J., Niu, N., Xu, S., Jin, Z. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+protocol+for+isolating+mouse+lung+endothelial+cells.">A simple protocol for isolating mouse lung endothelial cells.</a> Scientific Reports. 9 (1), 1458 (2019).</li><li>Wong, E., Nguyen, N., Hellman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+mouse+lung+endothelial+cells.">Isolation of primary mouse lung endothelial cells.</a> Journal of Visualized Experiments. (177), e63253 (2021).</li><li>Kraan, 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=Endothelial+CD276+(B7-H3)+expression+is+increased+in+human+malignancies+and+distinguishes+between+normal+and+tumour-derived+circulating+endothelial+cells.">Endothelial CD276 (B7-H3) expression is increased in human malignancies and distinguishes between normal and tumour-derived circulating endothelial cells.</a> British Journal of Cancer. 111 (1), 149-156 (2014).</li><li>Khan, S. Y., 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=Premature+senescence+of+endothelial+cells+upon+chronic+exposure+to+TNF&#945;+can+be+prevented+by+N-acetyl+cysteine+and+plumericin.">Premature senescence of endothelial cells upon chronic exposure to TNF&#945; can be prevented by N-acetyl cysteine and plumericin.</a> Scientific Reports. 7 (1), 39501 (2017).</li><li>Cossarizza, 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=Guidelines+for+the+use+of+flow+cytometry+and+cell+sorting+in+immunological+studies+(second+edition).">Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).</a> European Journal of Immunology. 49 (10), 1457 (2019).</li><li>Miron, R. J., Chai, J., Fujioka-Kobayashi, M., Sculean, A., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+24+protocols+for+the+production+of+platelet-rich+fibrin.">Evaluation of 24 protocols for the production of platelet-rich fibrin.</a> BMC Oral Health. 20 (1), 310 (2020).</li><li>Lenting, P. J., Christophe, O. D., Denis, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Willebrand+factor+biosynthesis,+secretion,+and+clearance:+Connecting+the+far+ends.">von Willebrand factor biosynthesis, secretion, and clearance: Connecting the far ends.</a> Blood. 125 (13), 2019-2028 (2015).</li><li>Thompson, S., Chesher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lot-to-lot+variation.">Lot-to-lot variation.</a> The Clinical Biochemist Reviews. 39 (2), 51-60 (2018).</li><li>Plebani, R., 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=Modeling+pulmonary+cystic+fibrosis+in+a+human+lung+airway-on-a-chip.">Modeling pulmonary cystic fibrosis in a human lung airway-on-a-chip.</a> Journal of Cystic Fibrosis. 21 (4), 606-615 (2021).</li></ol>

The multiple roles played by vascular endothelial cells in human pathophysiology make these cells an indispensable tool for in vitro pathogenetic and pharmacological studies. Since ECs from different vascular sites/organs display peculiar features and functions, the availability of healthy and diseased ECs from the organ of interest would be ideal for research purposes. For instance, HLMVECs are essential for studies on lung inflammation; therefore, a methodology for the high-yield, high-purity isolation of thes...

The vascular endothelium lines the inner surface of the blood vessels. It plays key roles in regulating blood coagulation, vascular tone, and immune-inflammatory responses1,2,3,4. Although the culture of endothelial cells (ECs) isolated from human specimens is essential for research purposes, it must be remarked that the ECs from different blood vessels (arteries, veins, capillaries) have specific functions. These cannot be fully recapitulated by human umbilical vein endothelial cells (HUVEC), which are easily available and widely used in studies on vascular endothelium pathophysiology5,6. For instance, human lung microvascular endothelial cells (HLMVECs) play key roles in lung inflammation by controlling leukocyte recruitment and accumulation4,7. Thus, an experimental setting aimed at reproducing lung inflammation with high fidelity should include HLMVECs. On the other hand, EC dysfunction can be observed in several pathologies; therefore, ECs from the patient are fundamental to building a reliable in vitro model of the disease. For instance, the isolation of fragments of ECs from the pulmonary artery (HPAECs), dissected from the explanted lungs of people affected by cystic fibrosis (CF), have enabled us to uncover mechanisms of endothelial dysfunction in this disease8,9.
Thus, protocols aimed at optimizing the isolation of ECs from different sources/organs also in disease states are essential to provide investigators with valuable research tools, particularly when these tools are not commercially available. HLMVEC and HPAEC isolation protocols have been previously reported10,11,12,13,14,15,16,17,18,19. In all cases, the enzymatic digestion of the lung specimens resulted in mixed cell populations, which were purified using ad hoc selective media and magnetic beads- or cytometric-based cell sorting. Further optimizations of these protocols must address two main issues in EC isolation: (1) cell and tissue contamination, which should be resolved at the earliest possible culture passages to minimize EC replicative senescence20; and (2) the low yield of primary EC isolates.
This study describes a new protocol for the high-yield, high-purity isolation of HLMVECs and HPAECs. This procedure can be easily applicable and give virtually pure macrovascular and microvascular ECs in a few steps.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% trypsin-EDTA 1X</td>
			<td>GIBCO</td>
			<td>25300-054</td>
			<td>Used to detach cells from the culture plates</td>
		</tr>
		<tr>
			<td>Anti CD31 Antibody, clone WM59</td>
			<td>Dako</td>
			<td>M0823</td>
			<td>Used for CD-31 staining in immunocytochemistry. Dilution used: 1:50</td>
		</tr>
		<tr>
			<td>Anti vWF Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MA5-14029</td>
			<td>Used for von Willebrand factor staining in immunocytochemistry. Working dilution: 1:100</td>
		</tr>
		<tr>
			<td>Autoclavable surgical scissors</td>
			<td>Any</td>
			<td></td>
			<td>Used for chopping specimens</td>
		</tr>
		<tr>
			<td>Cell strainers 40 &#181;m</td>
			<td>Corning</td>
			<td>431750</td>
			<td>Used during the second filtration</td>
		</tr>
		<tr>
			<td>Cell strainers 70 &#181;m</td>
			<td>Corning</td>
			<td>431751</td>
			<td>Using during the first filtration</td>
		</tr>
		<tr>
			<td>Collagenase, Type 2</td>
			<td>Worthington</td>
			<td>LS004177</td>
			<td>Type 2 Collagenase used for enzymatic digestion. Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Conjugated anti CD31 Antibody</td>
			<td>BD Biosciences</td>
			<td>555445</td>
			<td>Used for cell sorting (1:20 dilution)</td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS) with&#160; CaCl2 and MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td>Used for cell washing before medium change</td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS) without CaCl2 and MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Used for washing surgical specimens and cells before trypsinization</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium MV</td>
			<td>PromoCell</td>
			<td>C-22020</td>
			<td>HLMVEC growth medium</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td>Fibronectin from human plasma used for plate coating. Working concentration: 50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin, type A</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td>Used for plate coating</td>
		</tr>
		<tr>
			<td>Type A gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>g-2500</td>
			<td>Gelatin from porcine skin used for plate coating. Working concentration: 1.5%</td>
		</tr>
	</tbody>
</table>

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.

HLMEC isolation 
The main problem during the isolation of HLMVECs is the presence of contaminating cells since the microscopic capillaries cannot be easily separated from the stromal tissue. Therefore, achieving the highest possible purity at the earliest stages of the isolation process is crucial in order to reduce the culture passages and, thus, the cell aging. Likewise, an optimal isolation protocol should give the highest possible yield of pure HLMVECs. To achieve these goals, a new procedure wa...

Watch this Scientific Journal Video about Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples at JoVE.com

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

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 ...

This protocol is relevant for the isolation of endothelial cells from tissue samples or very small pieces of blood vessels. The main advantage of this technique is that it can be reproduced in any research laboratory and it allows to obtain a good purity of endothelial cells, even from very small samples. Begin preparing the lung parenchyma sample by washing the collected samples in a 50 milliliter tube containing PBS without calcium chloride and magnesium chloride, or PBS minus minus.Transfer the samples into a sterile Petri dish and chop manually into small fragments of about three to four millimeters using surgical scissors. For the artery segment, wash the collected samples in a 50-milliliter tube containing PBS minus minus, and transfer it into a sterile Petri dish without chopping. To remove the large part of residual blood, give two washes of PBS minus minus to the diced lung parenchyma or the pulmonary artery segment.Next, place the samples in 15 milliliter tubes and incubate with five milliliters of type II collagenase for 10 minutes at 37 degrees Celsius and 5%carbon dioxide. To remove the large fragments, place a sterile one-millimeter mesh-size strainer on the top of the 50 milliliter collection tube and pour the entire contents from the 15 milliliter tube onto the strainer. Next, gently massage the digested tissue with a spatula before rinsing the sample with PBS minus minus until the collection tube is filled.To remove nonvascular endothelial cells, filter the outflow collected from digested cells in a fresh 50-milliliter tube using a 70 micrometer mesh-size cell strainer. Then using a 40 micrometer mesh-size cell strainer, collect the outflow in a fresh 50 milliliter tube labeled as Tube 1. Centrifuge the samples at 30 G for five minutes at room temperature to sediment the cell clusters.Use a sterile pipette to transfer the supernatant to a fresh 50-milliliter tube labeled as Tube 2. Suspend the pellet in Tube 1 and fill the tube up to 50 milliliters with PBS minus minus. Similarly, fill Tube 2 up to 50 milliliters with PBS minus minus.Once done, centrifuge the samples at 300 G for five minutes at room temperature. Suspend the pellets with two milliliters of growth medium and seed the suspension in two separate wells of a pre-coded six-well plate. It was observed that part of the treated cell aggregates, including the smooth muscle cells or SMCs and fibroblasts, were bound to long fibers.Also, SMCs represented the majority of non-blood cell singlets. Small compact clusters of cells with diameters not exceeding 10 to 20 micrometers were without elements attributable to fragments of stromal tissue. Approximately 70%pure human lung microvascular endothelial cells, or HLMVEC, single cells were obtained.While obtaining pure HLMVEC preparations, the sorted cells assumed the characteristic endothelial-like shape. On focal microscopy showed that almost 100%of the fat's purified cells stained positive for endothelial cell antigens, namely CD31 and the more specific von Willebrand factor. When these cells were seated in matrigel, they generated tubular structures and HUVECs, confirming their endothelial phenotype.Due to the short incubation time during the enzymatic digestion, it is important to preheat the collagenase. And another point is that during the sedimentation of cell clusters, it is important to gently remove the supernatant without without touching the cell aggregates in the pallet. This procedure is based on the use of centrifugation and filtrations, and therefore could provide new insight to know to separate other cell types on the basis of their characteristics, such as size and clustering.

microvascular-macrovascular-endothelial-cell-isolation-purification

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1.8K vistas.  “G. d’Annunzio” University of Chieti-Pescara. Este protocolo es relevante para el aislamiento de células endoteliales de muestras de tejido o trozos muy pequeños de vasos sanguíneos. La principal ventaja de esta técnica es que se puede reproducir en cualquier laboratorio de investigación y permite obtener una buena pureza de las células endoteliales, incluso a partir de muestras muy pequeñas. Comience a preparar la muestra de parénquima pulmonar lavando las muestras recolectadas en un tubo de 50 mililitros que contenga PBS sin cl...