Name: Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples (Video) | JoVE Journal
Uploaded: 2023-02-03T14:00:00.000+00:00
Duration: 5 min 26 s
Description: Learn a reproducible protocol for isolating and purifying pulmonary microvascular and macrovascular endothelial cells from lung tissue using enzymatic digestion and filtration techniques. This method yields approximately 70% pure HLMVEC for studying lung inflammation and vascular biology.

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5 minutes

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 segment. Notably, the surgeons were accurately instructed to collect cancer-free samples. The presented protocol was optimized to obtain the greatest possible yield and purity.
1. Experimental preparation
<ol>
	<li>Collagenase reconstitution
	<ol>
		<li>Dissolve collagenase powder in phosphate-buffered saline without CaCl2 and MgCl2 (PBS&#8722;&#8722;, see Table of Materials) at a 2 mg/mL concentration, and filter the solution using a 0.22 &#181;m pore filter.</li>
		<li>Prepare 5 mL aliquots, and store them at &#8722;20 &#176;C. Thaw and preheat the aliquots to 37 &#176;C prior to use.</li>
	</ol>
	</li>
	<li>Plate coating
	<ol>
		<li>For plate coating, pipette 1.5% gelatin solution or 50 &#181;g/mL fibronectin (see Table of Materials) into the culture plate (500 &#181;L is enough to cover the surface of each well of a 6-well plate), and incubate for 1 h at 37 &#176;C.</li>
		<li>After incubation, remove the excess gelatin, and wash the wells with PBS&#8722;&#8722;. Aspirate the PBS&#8722;&#8722;, and let the plate dry in a sterile hood. 
		NOTE: For gelatin reconstitution, dissolve the powder in water to make a 1.5% solution, then autoclave it, and store at 4 &#176;C. Gelatin at 1.5% is not liquid at 4 &#176;C; therefore, it must be warmed up prior to plate coating. Alternatively, any commercial gelatin solution suitable for cell cultures can be used for the plate coating.</li>
	</ol>
	</li>
</ol>
2. Sample preparation
<ol>
	<li>Lung parenchyma
	<ol>
		<li>Wash the collected samples by immersing them in a 50 mL tube containing PBS&#8722;&#8722;.</li>
		<li>Transfer the samples into a sterile Petri dish, and manually chop the sample using surgical scissors (optimal size: &#62;2 cm) into small fragments of about 3-4 mm each.</li>
	</ol>
	</li>
	<li>Artery segment(s)
	<ol>
		<li>Wash the collected samples by immersing them in a 50 mL tube containing PBS&#8722;&#8722;.</li>
		<li>Transfer it into a sterile Petri dish without chopping, as fragmentation will increase the surface area of the cross-section of the artery segment, thus increasing the possibility of isolating non-ECs.</li>
	</ol>
	</li>
</ol>
3. Enzymatic digestion
<ol>
	<li>Wash the diced lung parenchyma or the pulmonary artery segment/s twice with PBS&#8722;&#8722;. This step will remove a large part of the residual blood.</li>
	<li>Place the samples in 15 mL tubes, and incubate with 5 mL of type 2 collagenase (see Table of Materials) for 10 min at 37 &#176;C and 5% CO2. During this incubation, the degradation of the extracellular matrix releases single cells and cell aggregates.</li>
</ol>
4. Recovery of the digested cells
<ol>
	<li>Place a sterile steel/metal strainer (i.e., a tea strainer with a ~1 mm mesh size) on the top of a 50 mL collection tube.</li>
	<li>Pour the entire contents from the 15 mL tube onto the strainer, gently massage the digested tissue with a spatula, and rinse the sample with PBS&#8722;&#8722;&#160;until the collection tube is filled. 
	&#8203;NOTE: This step will eliminate large tissue fragments on the millimeter scale, ensuring greater efficiency of the subsequent filtration steps.</li>
</ol>
5. Non-EC removal by filtration
<ol>
	<li>Filter the outflow collected in step 4.2 using a cell strainer with a 70 &#181;m mesh size, and collect the outflow in a fresh 50 mL tube.</li>
	<li>Then, use a cell strainer with a 40 &#181;m mesh size, and collect the outflow in a fresh 50 mL tube. Label this tube as &#34;tube 1&#34;. 
	&#8203;NOTE: These filtrations in step 5.1 and step 5.2 will remove large cell aggregates, which are often composed of mixed cell populations.</li>
</ol>
6. Sedimentation of cell clusters and cell seeding
<ol>
	<li>Centrifuge the samples at 30 x g for 5 min at room temperature to sediment the cell clusters.</li>
	<li>Carefully remove the supernatant using a sterile pipette (do not pour), and place it in a fresh 50 mL tube (&#34;tube 2&#34;).</li>
	<li>Suspend the pellet in &#34;tube 1&#34;, and fill the tube up to 50 mL with PBS&#8722;&#8722;. Fill &#34;tube 2&#34; up to 50 mL with PBS&#8722;&#8722;. 
	NOTE: From this step onward, both tubes are treated in the same way.</li>
	<li>Centrifuge the samples at 300 x g for 5 min at room temperature.</li>
	<li>Suspend the pellets with 2 mL of growth medium (see Table of Materials), and seed the suspension in two separate wells of a pre-coated 6-well plate (step 1.2). 
	&#8203;NOTE: From this step, feed the cells every 2 days with the growth medium until they reach confluency (approximately 1 week, depending on the sample size) in order to obtain a reasonable number of cells for sorting.</li>
</ol>
7. Cell expansion
<ol>
	<li>Wash the cells with 2 mL of PBS with CaCl2 and MgCl2 (PBS++, see Table of Materials) to remove the residual blood cells (using PBS++ is important to avoid cell detachment).</li>
	<li>Remove the PBS++, and add fresh culture medium.</li>
	<li>Repeat step 7.1 and step 7.2 until the cells reach confluency (approximately 1 week, depending on the sample size).</li>
</ol>
8. Cell sorting
<ol>
	<li>Detach the cells using 500 &#181;L of trypsin-EDTA (0.05%, see Table of Materials), centrifuge, and resuspend the pellet with 190 &#181;L of PBS&#8722;&#8722;.</li>
	<li>Add 10 &#181;L of a fluorescent conjugate anti-human CD31-FITC antibody (1:20 dilution, see Table of Materials), and incubate the cell suspension at 4 &#176;C for 30 min.</li>
	<li>Wash the cells using 10 mL of PBS&#8722;&#8722;, centrifuge at 300 x g for 5 min at room temperature, and resuspend the pellet in 300 &#181;L of PBS&#8722;&#8722;.</li>
	<li>Isolate and collect CD31 positive (CD31+) cells by fluorescence-activated cell sorting (FACS), using a 100 &#956;m nozzle, and collect them in a tube.
	<ol>
		<li>As per the gating strategy, first define the cell morphology using the side scatter area parameters, SSC-A and FSC-A. Then, select single cells using an FSC-Area/FSC-Height or SSC-Area/SSC-Height dot plot, define the positive cells in the marked sample versus the unstained control sample, and direct the fluorescent cells into the collection tube21.</li>
	</ol>
	</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min at room temperature, and suspend the pellet in 2 mL of growth medium for seeding in the pre-coated wells of a 6-well plate.</li>
</ol>
9. Post-sorting expansion
<ol>
	<li>Two days after cell sorting, replace the conditioned medium with fresh growth medium.</li>
	<li>Repeat step 7.1 and step 7.2 every 2 days for cell expansion.</li>
</ol>

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<li>Totani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+endothelial+cell+dysfunction+in+cystic+fibrosis%5BTitle%5D)+AND+%22Biochimica+Et+Biophysica+Acta.+Molecular+Basis+of+Disease%22%5BJournal%5D)">Mechanisms of endothelial cell dysfunction in cystic fibrosis.</a> Biochimica Et Biophysica Acta. Molecular Basis of Disease. 1863 (12), 3243-3253 (2017).</li>
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<li>Hewett, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+human+endothelial+cells+from+micro-+and+macro-vessels%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+endothelial+cells+from+fresh+tissues%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Human+primary+lung+endothelial+cells+in+culture%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+Cell+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+maintenance+of+human+pulmonary+artery+endothelial+cells+in+culture+isolated+from+transplant+donors%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+characterisation+of+human+pulmonary+microvascular+endothelial+cells+from+patients+with+severe+emphysema%5BTitle%5D)+AND+%22Respiratory+Research%22%5BJournal%5D)">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/pubmed?term=((Culture+of+pulmonary+artery+endothelial+cells+from+pulmonary+artery+catheter+balloon+tips%3A+considerations+for+use+in+pulmonary+vascular+disease%5BTitle%5D)+AND+%22The+European+Respiratory+Journal%22%5BJournal%5D)">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/pubmed?term=((A+simple+protocol+for+isolating+mouse+lung+endothelial+cells%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">A simple protocol for isolating mouse lung endothelial cells.</a> Scientific Reports. 9 (1), 1458(2019).</li>
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<li>Kraan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+CD276+%28B7-H3%29+expression+is+increased+in+human+malignancies+and+distinguishes+between+normal+and+tumour-derived+circulating+endothelial+cells%5BTitle%5D)+AND+%22British+Journal+of+Cancer%22%5BJournal%5D)">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>
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The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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><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 &amp;micro;m</td><td>Corning</td><td>431750</td><td>Used during the second filtration</td></tr><tr><td>Cell strainers 70 &amp;micro;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&amp;prime;s Phosphate Buffered Saline (PBS) with&amp;nbsp; CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>D8662</td><td>Used for cell washing before medium change</td></tr><tr><td>Dulbecco&amp;prime;s Phosphate Buffered Saline (PBS) without CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt;</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 &amp;micro;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

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 was ...

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 ...

microvascular-macrovascular-endothelial-cell-isolation-purification

Research

JoVE Journal

Biology

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Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolation of Primary Mouse Lung Endothelial Cells

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Murine Dermal Lymphatic Endothelial Cell Isolation

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

Isolation of Mouse Pancreatic Endothelial Cells

Learn a reproducible protocol for isolating and purifying pulmonary microvascular and macrovascular endothelial cells from lung tissue using enzymatic digestion and filtration techniques. This method yields approximately 70% pure HLMVEC for studying lung inflammation and vascular biology.

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

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