This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1. Isolation of Aorta from Mice
All the procedures described here were approved by the Institutional Animal Care and Use Committee of Wayne State University.
<ol>
	<li>Put the mouse into the induction chamber of the anesthesia machine. Set the isoflurane flow to 4% in conjunction with 25% fresh air and 75% O2. Anesthetize the mouse by inhalation of isoflurane (4% for induction, &#177; 1.0% for maintenance) in conjunction with 25% fresh air and 75% O2 via induction chamber followed by a dedicated nose cone. NOTE:&#160;Anesthesia with isoflurane can be delivered in 75% oxygen/25% air or in 100% oxygen.&#160;</li>
	<li>Check the mouse every 5 min until the appropriate level of anesthesia is reached (lack of withdrawal to toe pinch).</li>
	<li>Take the mouse out of the induction chamber and continue isoflurane inhalation though the dedicated nose cone that is connected to the anesthesia machine. Switch the isoflurane flow to 1% to maintain anesthesia.</li>
	<li>While it is under anesthesia, put the mouse onto a surgery pad, positioned on its back. Use laboratory tape to secure the limbs.</li>
	<li>Use a heating lamp to keep the mouse warm. Keep the lamp in an appropriate distance from the mouse so that the mouse will not over-heat.</li>
	<li>Spray the chest with 70% ethanol.</li>
	<li>Use dissection scissors to open the abdomen from the midline, and expose the abdominal aorta. Open the chest cavity and expose the heart and lungs.</li>
	<li>Cut the abdominal aorta at the middle with dissection scissors to release the blood.</li>
	<li>Fill a 1 mL syringe (with 25 G needle) with 1 mL of PBS containing 1,000 U/mL of heparin. Inject PBS containing 1,000 U/mL of heparin to the left ventricle and perfuse the aorta. Push the heart and the lung with forceps at the great arteries to the right side of the mice to expose the thoracic aorta.</li>
	<li>Quickly remove the thoracic aorta using micro-dissection forceps and put it in ice-cold 1x PBS (sterile), then transport the container into a laminar airflow hood.</li>
	<li>Insert a 1 mL syringe fitted with 25 G needle into one end of the aorta, and gently flush the aorta with ice-cold PBS to remove the blood.</li>
	<li>Use micro-dissection forceps to remove as much of the attached adipose tissue and small lateral vessels as possible.</li>
	<li>Immediately transfer the aorta to endothelial growth medium.</li>
	<li>Cut the aorta into 1 mm rings using a sterile scalpel blade. Harvest about 8 - 10 rings per aorta.</li>
	<li>Open each aortic ring using a pair of micro-dissection scissors.</li>
</ol>
2. Seed the Aortic Segments on Matrix
<ol>
	<li>When not in use, keep the growth factor-reduced matrix in -20 &#176;C to prevent solidification. Put the growth factor-reduced matrix in 4 &#176;C at least overnight to allow a complete thaw.</li>
	<li>Precool a 6-well plate and pipet tips to -20 &#176;C for at least 10 min. Put the 6-well plate on ice and coat one well of the plate with 1 mL of matrix without introducing any air bubbles. Place the plate in a 37 &#176;C incubator for 20 min to allow the matrix to solidify.</li>
	<li>Implant the aortic pieces onto the solidified matrix using sterile micro-dissection forceps. Place the pieces lumen-side-down on the matrix without touching the endothelium. Place 3 - 4 aortic segments close to each other on the matrix.</li>
	<li>Add just enough endothelial cell growth medium to keep segments wet (~ 200 &#181;L).</li>
	<li>Incubate the plate at 37 &#176;C under 5% CO2 for 4 to 6 h. At the end of the day, add just enough medium to cover the aortic segments.</li>
	<li>Observe the aortic segment sprouting periodically under a phase-contrast microscope. Check medium level and cell growth each day. Add medium if necessary to keep aortic segments covered.</li>
	<li>On the 4th day, gently remove the medium and remove the aortic segments from the matrix using a sterile needle without interrupting the growing endothelial cells.</li>
	<li>Add 2 mL of new endothelial cell growth medium and allow the endothelial cells continue to proliferate on matrix for 2 - 3 days.</li>
</ol>
3. Initial Passaging of the Mouse Aortic Endothelial Cells
<ol>
	<li>Coat a new T12.5 flask with gelatin (0.1%), incubate at 37 &#176;C for 30 min.</li>
	<li>Wash the matrix plates carefully with sterile 37 &#176;C PBS.</li>
	<li>Add 2 mL of neutral proteinase (50 U/mL) to the matrix plates and incubate at room temperature on platform rocker with occasional shaking. Check the cells under a phase-contrast microscope to make sure the majority of cells are detached.</li>
	<li>Add 2 mL of D-Val to inactivate the neutral proteinase.</li>
	<li>Collect the supernatant carefully in a 15 mL centrifuge tube. Wash the plate with 2 mL D-Val and collect the supernatant.</li>
	<li>Centrifuge the cell suspension at 900 x g for 5 min at room temperature.</li>
	<li>Re-suspend the cell pellet in 4 mL of endothelial cell growth medium and plate in the T12.5 flask coated with gelatin. Incubate the cells at 37 &#176;C, 5% CO2 for 2 h.</li>
	<li>Replace the medium. Incubate the cells at 37 &#176;C, 5% CO2 until they are 85% - 90% confluent.</li>
</ol>
4. Passaging of the Mouse Aortic Endothelial Cells
<ol>
	<li>Coat two new T12.5 flasks with gelatin (0.1%), incubate at 37 &#176;C for 30 min. Pre-heat Trypsin-EDTA (0.25% Trypsin, 0.02 EDTA in PBS) and sterile PBS at 37 &#176;C for about 15 min.</li>
	<li>Wash the cells carefully with sterile 37 &#176;C PBS.</li>
	<li>Add 0.5 mL of Trypsin-EDTA (0.25% Trypsin, 0.02 EDTA in PBS) and incubate at 37 &#176;C for ~ 1 min or until the majority of the cells turn round.</li>
	<li>Add 2 mL of endothelial cell growth medium to stop the digestion. Pipette the cell suspension up and down within the flask several times. If necessary, use a cell scraper to collect the cells.</li>
	<li>Collect the cell suspension in a 15 mL centrifuge tube. Centrifuge the cell suspension at 900 x g for 5 min at room temperature.</li>
	<li>Re-suspend the cell pellet in 4 mL of endothelial cell growth medium and plate in 2 T12.5 flasks coated with gelatin. Incubate the cells at 37 &#176;C, 5% CO2 for 2 h.</li>
	<li>Replace the medium. Incubate the cells at 37 &#176;C, 5% CO2 until they are 85% - 90% confluent.</li>
	<li>Use the mouse aortic endothelial cells after 2 - 3 passages.</li>
</ol>
5. Characterization of the Mouse Aortic Endothelial Cells
<ol>
	<li>Re-plate the cells.
	<ol>
		<li>Coat a 6-well plate with gelatin (0.1%), incubate at 37 &#176;C for 30 min. Rinse the plate with PBS.</li>
		<li>Pre-heat the Trypsin-EDTA and sterile PBS to 37 &#176;C for about 15 min.</li>
		<li>Wash the cells with sterile PBS 3 times. Add 0.5 mL of Trypsin/EDTA to the cells, incubate for ~1 min at 37 &#176;C or until the majority of the cells turn round.</li>
		<li>Add 2 mL of endothelial cell growth medium to stop the digestion. Pipette the cell suspension up and down within the flask several times. If necessary, use a cell scraper to collect the cells.</li>
		<li>Collect the cell suspension in a 15 mL centrifuge tube. Centrifuge the cell suspension at 900 x g for 5 min at room temperature.</li>
		<li>Discard the supernatant, re-suspend the cells in endothelial cell growth medium. Seed the cells in 6-well plate coated with gelatin at the density of 3 x 105/well.</li>
		<li>Incubate the cells overnight at 37 &#176;C, 5% CO2 to allow the cells to attach to the culture surface.</li>
	</ol>
	</li>
	<li>Dil-ac-LDL uptaken and Ulex-lectin binding.
	<ol>
		<li>Store the 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL) at -20 &#176;C. Before staining, thaw Dil-ac-LDL on ice. The stock Dil-ac-LDL solution is 1 mg/mL. Store the FITC-labeled Ulex europaeus agglutinin (Ulex-Lectin, 1 &#181;g/mL) at 4 &#176;C. The stock Ulex-Lectin solution is 1 mg/mL. Store Hoechst 33342 at 4 &#176;C. The stock Hoechst 33342 solution is 100 &#181;g/mL.</li>
		<li>Add 10 &#181;L of Dil-ac-LDL stock solution to 1 mL of culture medium to make a final concentration of 10 &#181;g/mL.</li>
		<li>Prior to staining, remove the old medium and wash the cells in each well with new medium.</li>
		<li>Remove the new medium and add the Dil-ac-LDL staining medium.</li>
		<li>Incubate the cells at 37 &#176;C, 5% CO2 for 1 h.</li>
		<li>Wash the cells with sterile 1x PBS 3 times.</li>
		<li>Add 1 mL of 10% formalin to fix the cells. Put the plate in room temperature for about 30 min.</li>
		<li>Wash the cells with sterile 1x PBS 3 times. Avoid light.</li>
		<li>Add 1 mL sterile 1x PBS that contains 10 &#181;L of Ulex-Lectin.</li>
		<li>Incubate the cells at room temperature in the dark for 1 h.</li>
		<li>Remove the Ulex-Lectin by washing the cells with sterile 1x PBS 3 times.</li>
		<li>Add Hoechst 33342 to final concentration of 1 &#181;g/mL, incubate 10 min in the dark.</li>
		<li>View the fluorescence of Dil-ac-LDL (red, excitation wavelength of 576 nm), Ulex-Lectin (green, excitation wavelength of 519 nm) and Hoechst (blue, excitation wavelength of 361 nm) with an inverted fluorescent microscope.</li>
	</ol>
	</li>
	<li>Fluorescent staining for CD31, VEGFR2, eNOS, VE-Cadherin and Calponin.
	<ol>
		<li>Store FITC-conjugated anti-mouse CD31 antibody, eNOS antibody, VE-Cadherin antibody, FITC-conjugated anti-rabbit IgG at 4 &#176;C in the dark. Store VEGFR2 antibody in -20 &#176;C.</li>
		<li>Wash the cells with sterile 1x PBS 3 times.</li>
		<li>Add 1 mL of 10% formalin to fix the cells. Put the plate in room temperature for about 30 min.</li>
		<li>Wash the cells with sterile 1x PBS 3 times.</li>
		<li>To stain CD31, add 1 mL of sterile 1x PBS that contains 10 &#181;L of CD31-FITC antibody. To stain either VEGFR2, eNOS, VE-Cadherin or calponin, add 1 mL of sterile 1x PBS that contains 4 &#956;L of VEGFR2, eNOS, VE-Cadherin or calponin primary antibody, respectively.</li>
		<li>Incubate the cells on ice for 1 h in the dark.</li>
		<li>Remove the antibodies by washing the cells with sterile PBS 3 times.</li>
		<li>For the staining of VEGFR2, eNOS, VE-Cadherin or calponin, add 1 mL of sterile 1x PBS that contains 10 &#956;L of FITC-conjugated anti-rabbit IgG. Incubate the cells on ice for 1 h in the dark. Remove the IgG by washing the cells with sterile PBS 3 times.</li>
		<li>Add Hoechst 33342 to final concentration of 1 &#181;g/mL, incubate 10 min in the dark.</li>
		<li>View the fluorescence of CD31, VEGFR2, eNOS, VE-Cadherin, calponin (green, excitation wavelength of 518-535 nm) and Hoechst (blue, excitation wavelength of 361 nm) with an inverted fluorescent microscope.</li>
	</ol>
	</li>
</ol>

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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=C-Reactive+protein-induced+endothelial+microparticle+generation+in+HUVECs+is+related+to+BH4-dependent+NO+formation.">C-Reactive protein-induced endothelial microparticle generation in HUVECs is related to BH4-dependent NO formation.</a> Journal of vascular research. 44, 241-248 (2007).</li><li>Wang, J. 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=Increased+circulating+CD31+/CD42-+microparticles+are+associated+with+impaired+systemic+artery+elasticity+in+healthy+subjects.">Increased circulating CD31+/CD42- microparticles are associated with impaired systemic artery elasticity in healthy subjects.</a> American journal of hypertension. 20, 957-964 (2007).</li><li>Mackay, L. 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The authors have nothing to disclose.

This study demonstrates a simple method to isolate and culture endothelial cells from a mouse aorta without any special equipment. The immunofluorescence staining indicated that the majority of cells were endothelial cells after the second passage. It is suggested that cells at second to third passage are suitable for in vitro and in vivo experiments to study endothelial biology.
The Key Notes from the Present Protocol
There are fi...

The vascular endothelium is not only a barrier layer that separates blood and tissue, it is considered a vast endocrine gland that stretches over the entire vascular tree with a surface area of 400 square meters1. The well-being of the endothelium is essential to vascular homeostasis. The dysfunctional endothelium participates in various cardiovascular disorders, including atherosclerosis, vasculitis and ischemia/reperfusion injuries, etc. 2-4. To date, the specific cellular and molecular mechanisms involved in these disease settings are not well understood due to the diffused anatomic nature of endothelium.
The mouse is an important model for research because genetic manipulation techniques are more developed in mice than in any other mammalian species. However, the isolation of primary murine aortic endothelial cells is considered particularly difficult because the small size of the aorta makes enzymatic digestion of endothelium impractical. Some reported procedures to isolate and purify ECs require 5-7.
The goal of this protocol is to use a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. In this protocol, the freshly isolated aorta is cut into small segments and seeded onto a matrix with the endothelium facing down to allow for endothelial sprouting. After segments are removed, endothelial cells are expanded in endothelium-favored medium and are ready for experiments after two or three passages. The advantages of the described method are that: 1) considerably high numbers of endothelial cells are harvested from a single aorta; 2) cell viability is well preserved; and 3) no special equipment or technique is needed. It provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell pathophysiology. For those who are interested in studying primary cultured endothelial cells from either gene knock-out mice, gene knock-in mice, or a murine disease model, this protocol is very useful and easy to practice.

<table><tbody><tr><td>4- or 6-week-old mice</td><td>Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Sterile 1x phosphate-buffered saline (PBS)</td><td>Gibco</td><td>#10010-023</td><td></td></tr><tr><td>Sterile 1x PBS containing 1,000 U/mL of heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Endothelial cell growth medium (Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium [DMEM] with 25&amp;nbsp;mM HEPES [Gibco, #12320-032], supplemented with 100&amp;nbsp;&amp;mu;g/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000&amp;nbsp;U/mL heparin [Sigma Aldrich, H3149], 10,000&amp;nbsp;U/mL penicillin and 10&amp;nbsp;mg/mL streptomycin [Gibco, #15140-122])</td><td></td><td></td><td></td></tr><tr><td>Growth factor reduced matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Neutral proteinase (Dispase, 1&amp;nbsp;U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034&amp;nbsp;g/L, Sigma, #1255), in Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium, low glucose (Gibco, #12320-032)</td><td></td><td></td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)</td><td>Life Technologies</td><td>L3484</td><td></td></tr><tr><td>FITC-labeled &lt;em&gt;Ulex europaeus&lt;/em&gt; agglutinin (&lt;em&gt;Ulex&lt;/em&gt;-Lectin)</td><td>Sigma</td><td>L9006</td><td></td></tr><tr><td>Anti-mouse CD31-FITC conjugated antibody</td><td>BD Biosciences</td><td>553372</td><td></td></tr><tr><td>Anti-mouse vascular endothelial growth factor receptor 2 antibody</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Anti-mouse endothelial nitric oxide synthase</td><td>Abcam</td><td>ab5589</td><td></td></tr><tr><td>Anti-mouse vascular endothelium-cadherin</td><td>Abcam</td><td>33168</td><td></td></tr><tr><td>Anti-mouse calponin</td><td>Abcam</td><td>700</td><td></td></tr><tr><td>FITC-conjugated anti-rabbit IgG</td><td>Sigma</td><td>F6005</td><td></td></tr><tr><td>1 mL syringe fitted with 25-G needle</td><td>Fisher Scientific</td><td>50-900-04222</td><td></td></tr><tr><td>100 mm Peri dishes</td><td>Fisher Scientific</td><td>07-202-516</td><td></td></tr><tr><td>Six-well cell culture plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>T12.5 culture flask</td><td>Fisher Scientific</td><td>50-202-076</td><td></td></tr><tr><td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade</td><td>Fine Science Tools, Inc.</td><td></td><td></td></tr><tr><td>Anesthesia machine with isoflurane</td><td>Webster Veterianary Supply</td><td>07-806-3204</td><td></td></tr><tr><td>heating lamp</td><td></td><td></td><td></td></tr><tr><td>Centrifuge machine</td><td></td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td></td><td></td><td></td></tr><tr><td>inverted fluorescence microscope</td><td></td><td></td><td></td></tr></tbody></table>

isolation and primary culture of mouse aortic endothelial cells

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

Endothelial Cell Sprouting
Spontaneous endothelial cell sprouting started from a mouse aorta segment. The mouse aorta segment was allowed to grow on a growth factor-reduced matrix then in endothelial cell growth medium for 4 days. The endothelial cell sprouting usually appears in 2 - 4 days. Photomicrographs were taken on day 4 (Figure 1). As shown in the pictures, numerous endothelial cells migrate away from the segment. The newly formed sprouts continue to e...

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Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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

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

isolation-and-primary-culture-of-mouse-aortic-endothelial-cells

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30.4K Views.  Wayne State University. The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Video: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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 and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Medicine

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Isolation of Embryonic Ventricular Endothelial Cells

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

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

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

Isolation of 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 ...

Immunology and Infection

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Mouse Coronary Endothelial Cells

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

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

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

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

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

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

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

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...