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 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.
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.
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.
2. Seed the Aortic Segments on Matrix
3. Initial Passaging of the Mouse Aortic Endothelial Cells
4. Passaging of the Mouse Aortic Endothelial Cells
5. Characterization of the Mouse Aortic Endothelial Cells
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 extend from the segment and the branch.
Endothelial Cell Phenotypes
These cells demonstrated spindle-shaped and cobblestone-like appearances after initial passage (Figure 2A). The attached cells were labeled with Dil-ac-LDL and Ulex-Lectin for one hour. As shown in Figures 2B-2E, most of the cells were double-positive for Dil-ac-LDL uptake (red) and Ulex-Lectin binding (green). Meanwhile, after the second passage, more than 95% of the cells were positive for platelet endothelial cell adhesion molecule 1 (CD31, PECAM-1, Figure 2F), VEGFR2 (Figure 2G), VE-Cadherin (Figure 2H), eNOS (Figure 2I) but negative for Calponin (Figure 2J) which is a smooth muscle cell marker.
Figure 1. Endothelial Cell Sprouting. Mouse aortic segment was seeded onto growth factor-reduced matrix and cultured in endothelial cell growth medium. Endothelial cell sprouting started as early as on day 2 (A, bar = 100 µm) and increased in the following 2 – 3 days (B, C, bar = 100 µm). The segment was removed on day 4 (D, bar = 500 µm).
Figure 2. Endothelial Cell Phenotypes. The attached cells display spindle-shaped and cobblestone-like appearances (A, bar = 100 µm), double-positive fluorescence of Dil-ac-LDL and Ulex-Lectin (B-E, bar = 100 µm). Meanwhile, platelet endothelial cell adhesion molecule 1 (CD31, PECAM-1), VEGFR2, VE-Cadherin, eNOS were positive in >95% of the cells after the second passage (F-I, bar = 100 µm) Most of the cells were negative for Calponin staining, which is a smooth muscle cell marker (J, bar = 100 µm).
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 five critical points in the procedure. First, the vascular lumen is flushed with PBS containing heparin to minimize endothelial cell activation and clot formation. Second, the time between cardiac arrest and the seeding of aortic segment onto matrix is critical to endothelium viability. While clearing the peri-arterial adipose tissue and connective tissue, avoid stretching the aorta and limit the time spent on this step to 10 – 15 min. Third, pour just enough media to cover the segments after they are seeded. Too much media will cause the aortic segments to float. Fourth, the culture medium contains a high concentration of endothelial cell growth supplement, making endothelial cells grow much faster than when cultured in endothelial growth medium-2. The outgrowth of endothelial cells will suppress other cell types such as smooth muscle cells and fibroblasts. Fifth, the timing to remove aortic segment from matrix is also critical to the purity of endothelial cells. This step prevents contamination by fibroblasts and smooth muscle cells. The segment should be removed before the development of the tube network. Delayed removal of aortic segment will result in contamination of other cell types such as fibroblasts or smooth muscle cells. Please note that this approach is also used to study angiogenesis in vitro and the formation of capillary like structures indicates angiogenesis capacity of the aorta segment. Based on our experience, if the segments are removed after the capillary-like structure fully develops, the incidence of smooth muscle cells contamination increases dramatically. Therefore, we feel that the best time point to remove the segment is when the network starts to be visible but is not fully developed. In this way, we are able to harvest as many endothelial cells as possible while keeping contamination by smooth muscle cells to a minimum.
The Limitations of the Present Protocol
There are some limitations of the described methods. First, there are chances of contamination of fibroblasts and smooth muscle cells, if the aortic segments are not removed before tube networks develop. The fibroblasts or smooth muscle cells may start to attach to the matrix and proliferate 3 to 5 days after seeding. Therefore, always remove the aortic segments in a timely manner. As a secondary precaution, changing the medium 2 hours after re-plating the cells onto a new flask also helps to eliminate the possible contamination of fibroblasts and smooth muscle cells. Second, these cells are cultured in vitro for 7 – 10 days after isolation. It is possible that their phenotypes may be different from freshly isolated endothelial cells. For example, if the endothelial cells from a mouse model of hyperlipidemia are cultured in endothelial cell growth medium without a high concentration of lipids, they are not exposed to the hyperlipidemic environment as they were in animals. The cultured endothelial cells may behave differently from freshly isolated endothelial cells8. This problem exists in all in vitro cultured primary cells and cell lines. Adding the pathological stimuli into the culture medium to mimic the in vivo environment may be a solution.
Endothelial Cells Demonstrate Different Phenotypes in Different Vascular Branches
The vascular system is a hierarchical structure composed of arteries, veins, and capillaries. The heterogeneity of endothelial cells that reside in the specific 'zones' of vasculature plays a large part in creating functional diversity in the vascular system9,10. Even in a single vascular bed, such as the aorta, endothelial heterogeneity exists. Laminar blood flow with high shear stress in the straight part of the aorta induces endothelial nitric oxide synthase and thrombomodulin11. Therefore, endothelial cells that resides in the straight part of the aorta demonstrate anti-coagulant, anti-adhesive, and anti-inflammatory properties12,13. In contrast, turbulent blood flow at areas where arteries branch or turn sharply (aortic arch) reduces endothelial nitric oxide synthase expression and induces atherogenic genes in endothelial cells, i.e., the monocyte chemotactic protein-1 (MCP-1), and platelet-derived growth factors (PDGFs), resulting in a pro-inflammatory and atherosclerotic endothelial phenotype6. In addition, endothelial cells in the vessels of each organ undergo anatomic and functional adaptive changes that are specific to that organ’s functions14,15 . Nonetheless, there is a consensus that several cell surface markers, functional genes and cell activities can be used to characterize endothelial cell lineage. These cell surface markers include Platelet endothelial cell adhesion molecule (PCAM-1, CD31), von Willebrand factor (vWF), Vascular Endothelial-Cadherin (VE-Cadherin, CD144). The most frequently used functional genes include endothelial nitric oxide synthase (eNOS) and Vascular Endothelial Growth Factor Receptor (VEGFR). The cell activities that are usually seen in endothelial cell lineage include uptake of Dil-ad-LDL and binding Lectin, which forms cord-like structures on the matrix.
The Significance and Future Applications of Primary Cultured Mouse Aortic Endothelial Cells
The method described here is used to study macrovascular endothelial cells. The significance of this protocol is that it provides a great opportunity to study the endothelial-specific activities of targeted molecules and can be done in knockout and transgenic mouse models, making it very useful in cardiovascular research. The ability to grow high numbers of mouse aortic endothelial cells under defined conditions makes this ideal technique to better define the phenotypes and functions of endothelium. This technique improves the practicality of testing the prospective potential of endothelial cell-based therapy in murine models, through either intravenous injection or engraftment of endothelial cells.
The authors have nothing to disclose.
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.
4- or 6-week-old mice (Jackson Laboratory, #000664). |
Sterile 1X phosphate-buffered saline (PBS, Gibco, #10010-023). |
Sterile 1X PBS containing 1,000 U/ml of heparin (Sigma Aldrich, H3149). |
Endothelial cell growth medium (Dulbeccos’ Modified Eagle’s Medium[DMEM] with 25mM HEPES[Gibco, #12320-032 ], supplemented with 100μg/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]). |
Growth factor reduced matrix (BD Biosciences, #356231). |
Neutral proteinase (Dispase, 1U/ml, Fisher Scientific #CB-40235) and D-Val medium (D-Valine, 0.034g/L[Sigma, #1255], in Dulbeccos’ Modified Eagle’s Medium, low glucose[Gibco, #12320-032 ]). |
Gelatin (100‑200 μg/cm2, Sigma, #G1393). |
1,1`-dioctadecyl-3,3,3`,3`- tetramethylindo- carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL, Life Technologies, #L3484), FITC-labeled Ulex europeus agglutinin (Ulex-Lectin, Sigma, #L9006), Anti-mouse CD31-FITC conjugated antibody (BD Biosciences, # 553372). |
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) |
One ml syringe fitted with 25-G needle (Fisher Scientific, #50-900-04222). |
100mm Peri dishes (Fisher Scientific, #07-202-516). |
Six-well cell culture plates (Fisher Scientific, #08-772-1B). |
T12.5 cultuer flask (Fisher Scientific, #50-202-076) |
Scissors, forceps, microdissection scissors and forceps, Scalpel blade (Fine Science Tools, Inc) |
Anesthesia machine with isoflurane (Webster Veterianary Supply, #07-806-3204), heating lamp |
Centrifuge machine. |
Inverted phase-contrast microscope. |
inverted fluorescence microscope. |