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>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. 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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

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 ...

microvascular-macrovascular-endothelial-cell-isolation-purification

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Biology

2.0K Views.  “G. d’Annunzio” University of Chieti-Pescara. Dit protocol is relevant voor de isolatie van endotheelcellen uit weefselmonsters of zeer kleine stukjes bloedvaten. Het belangrijkste voordeel van deze techniek is dat het in elk onderzoekslaboratorium kan worden gereproduceerd en het maakt het mogelijk om een goede zuiverheid van endotheelcellen te verkrijgen, zelfs uit zeer kleine monsters. Begin met het voorbereiden van het longparenchymmonster door de verzamelde monsters te wassen in een buis van 50 milliliter met PBS zonder calciumchlor...

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

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

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Cancer Research

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Immunology and Infection

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Neuroscience

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

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

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

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

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

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

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

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

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

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

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

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

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

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.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Isolation of Mouse Coronary Endothelial Cells

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

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

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

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

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

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.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

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

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