Name: isolation and primary culture of mouse aortic endothelial cells
Uploaded: 2016-12-19T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

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.
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	<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 induct...

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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.

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			<td>4- or 6-week-old mice (Jackson Laboratory, #000664).</td>
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			<td>Sterile 1X phosphate-buffered saline (PBS, Gibco, #10010-023).</td>
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			<td>Sterile 1X PBS containing 1,000 U/ml of heparin (Sigma Aldrich, H3149).</td>
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			<td>Endothelial cell growth medium (Dulbeccos&#8217; Modified Eagle&#8217;s Medium[DMEM] with 25mM HEPES[Gibco, #12320-032 ], supplemented with 100&#956;g/ml endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma,#2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/ml heparin [Sigma Aldrich, H3149], 10,000U/ml penicillin and 10mg/ml streptomycin [Gibco, #15140-122]).</td>
		</tr>
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			<td>Growth factor reduced matrix (BD Biosciences, #356231).</td>
		</tr>
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			<td>Neutral proteinase (Dispase, 1U/ml, Fisher Scientific #CB-40235) and D-Val medium (D-Valine, 0.034g/L[Sigma, #1255], in Dulbeccos&#8217; Modified Eagle&#8217;s Medium, low glucose[Gibco, #12320-032 ]).&#160;</td>
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			<td>Gelatin (100&#8209;200 &#956;g/cm2, Sigma, #G1393).</td>
		</tr>
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			<td>1,1`-dioctadecyl-3,3,3`,3`- tetramethylindo- carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL, Life Technologies, #L3484),&#160; FITC-labeled Ulex europeus agglutinin (Ulex-Lectin, Sigma, #L9006),&#160; Anti-mouse CD31-FITC conjugated antibody (BD Biosciences, # 553372).</td>
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			<td>Anti-mouse vascular endothelial growth factor receptor 2 antibody (Cell signaling, #9698), anti-mouse endothelial nitric oxide synthase (Abcam, #ab5589), anti-mouse vascular endothelium-cadherin (Abcam, #33168), anti-mouse calponin (Abcam, #700), FITC-conjugated anti-rabbit IgG (Sigma, #F6005)</td>
		</tr>
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			<td>One ml syringe fitted with 25-G needle (Fisher Scientific, #50-900-04222).&#160;</td>
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			<td>100mm Peri dishes (Fisher Scientific,&#160; #07-202-516).</td>
		</tr>
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			<td>Six-well cell culture plates (Fisher Scientific, #08-772-1B).</td>
		</tr>
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			<td>T12.5 cultuer flask (Fisher Scientific, #50-202-076)</td>
		</tr>
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			<td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade (Fine Science Tools, Inc)</td>
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			<td>Anesthesia machine with isoflurane (Webster Veterianary Supply, #07-806-3204), heating lamp</td>
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			<td>Centrifuge machine.</td>
		</tr>
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			<td>Inverted phase-contrast microscope.</td>
		</tr>
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			<td>inverted fluorescence microscope.</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...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

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 ...

The overall goal of this procedure is to use a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Other methods used to isolate endothelial cells are mostly applied to the heart or the lung, not the aorta. This technique for the aorta provides considerably higher yield, high cell viability, and requires no special equipment or techniques.While those apply into the studying of primary endothelial cells from either doing knockout or murine mice are from most disease models. This protocol is very useful and easy to practice. Start this protocol at the beginning of the day.After anesthetizing a mouse and confirming a lack of reflex, place the mouse supine on a surgical pad and position a heating lamp over it to maintain its body temperature. Then sanitize the abdomen by spraying it down with 70%ethanol. Next, with dissection scissors, open the abdomen along the midline to expose the abdominal aorta.Then, open the chest cavity to expose the heart and lungs. Now, release the blood from the mouse by dissecting along the center line of the aorta. Then, into the left ventricle, inject 1000 units of heparin and a milliliter of PBS, thus perfusing the aorta.After the perfusion, push aside impeding organs, quickly remove the thoracic aorta, and transfer it to ice cold PBS. Under a laminar airflow hood, gently flush the aorta with ice cold PBS to remove any remaining blood using a 25-gauge needle and syringe. Then, using microdissection forceps, remove as much attached adipose tissue and lateral vessels as possible.The time between cardiac arrest and the seeding of the aortic segment onto the matrix is critical to the endothelium's viability. So, while removing the periarterial adipose tissue and connective tissue, avoiding stretching the aorta, and limit the time spent on this step, too. At most, 15 minutes.Then, transfer the cleaned-off aorta to endothelial growth medium and cut the aorta into one-millimeter rings with a scalpel. One aorta yields up to 10 rings. Then, open each ring with microdissection scissors.To begin, put a six-well plate on ice and coat it with one milliliter of matrix solution without pouring out any air bubbles. Then, transfer the plate to an incubator for 20 minutes so the matrix solidifies. Once solidified, the aorta pieces can be placed on the matrix, lumen side down.Do so without touching the endothelium. Position three to four segments in each well. Then, pour just enough growth medium over the segments to keep them wet.Next, transfer them to an incubator with 5%carbon dioxide for about four to six hours. At the end of the day, add more medium so the segments are covered. Over the next several days, the aortic segments will periodically sprout.Monitor the sprouting by phase contrast microscopy every day. Adjust the level of medium at the same time. On the fourth day, carefully remove the medium, and gently remove the aortic segments.Do not disturb the cells growing on the matrix. Then, add two milliliters of fresh medium to the endothelial cells on the matrix, and continue the culturing for two to three days. The timing to remove aortic segments from the matrix is critical to the purity of the endothelial cells.The segment must be removed before the development of the tube network, or the cells will be contaminated with fibroblasts or smooth muscle cells. To begin, prepare a T12.5 flask with 0.1%gelatin, and incubate it for 30 minutes. Next, wash the matrix plates carefully with warmed PBS, then add 100 units of neutral protease to each well, and incubate the plates at room temperature on a platform rocker with occasional shaking.When the cells appear detached under phase contrast, add two milliliters of d-Val to each well to end the reaction. Examine the wells, and then collect the supernatants in a 15-milliliter tube. Then, centrifuge the tube at 900 gs for five minutes, and re-suspend the pellet of cells in four milliliters of medium.Plate the suspension in the prepared flask, and incubate the cells for two hours. Then, replace the medium, and continue the incubation for three to four days until the cells are 85-90%confluent. When the cells are ready, prepare two more T12.5 flasks to passage the cells as before.Then, wash the cells with warmed PBS, and use about half a milliliter of Trypsin-EDTA to detach the majority of them within a minute of incubation. Next, end this reaction with two milliliters of media. Now, flow the suspension through a pipette several times, and transfer it to a 15-milliliter tube.Next, centrifuge the cells at 900 gs for five minutes, and re-suspend the pellet in four milliliters of medium. Then, split the suspension between the two prepared flasks, and let them incubate for two hours. Then, replace the medium, and continue growing the cells to confluency as before.After two to three such passages, characterize the cells. Using the described protocol, spontaneous endothelial cell sprouting was observed for mouse aorta segments after two days in culture. After four days, numerous endothelial cells migrated away from the segment, and newly form sprouts continued to extend from the segment and the branch.After culturing these cells and passaging them once, the cells were spindle-shaped and cobblestone-like in appearance. The attached cells were then labeled with Dil-ac-LDL and Ulex Lectin for an hour. Nuclei were also stained.Most of the cells were double positive for Dil-ac-LDL uptake and Ulex binding. After the second passage, more than 95%of the cells were positive for platelet endothelial cell adhesion molecule one. The cells were also positive for VEGFR2, VE-Cadherin, and eNOS.Also, the cells were mostly negative for the smooth muscle marker calponin. This protocol provides a great opportunity to study the endothelial-specific activities of targeted molecules, and can be done in the knockout and transgenic mouse models, making it very useful in the field of cardiovascular research.

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