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Development of immortalized mouse aortic endothelial cell lines.

Ni CW, Kumar S, Ankeny CJ, Jo H - (2014)

Bottom Line: Here, we developed an effective method to prepare immortalized MAEC (iMAEC) lines.Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen.Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wallace H, Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, 1760 Haygood Drive, Health Science Research Building, E-170, Atlanta, GA 30322, USA. hanjoong.jo@bme.gatech.edu.

ABSTRACT

Background: The understanding of endothelial cell biology has been facilitated by the availability of primary endothelial cell cultures from a variety of sites and species; however, the isolation and maintenance of primary mouse aortic endothelial cells (MAECs) remain a formidable challenge. Culturing MAECs is difficult as they are prone to phenotypic drift during culture. Therefore, there is a need to have a dependable in vitro culture system, wherein the primary endothelial cells retain their properties and phenotypes.

Methods: Here, we developed an effective method to prepare immortalized MAEC (iMAEC) lines. Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen. Immortalized cells were then incubated with DiI-acetylated-low density lipoprotein and sorted via flow cytometry to isolate iMAECs.

Results: iMAECs expressed common markers of endothelial cells, including PECAM1, eNOS, VE-cadherin, and von Willebrand Factor. iMAECs aligned in the direction of imposed laminar shear and retained the ability to form tubes. Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.

Conclusion: In summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.

No MeSH data available.


Related in: MedlinePlus

Morphology of mouse aortic endothelial cells. (A) Aortic explants were cultured on top of collagen gel beads for 4 days. EC grew and migrated out of the aorta piece. (B) EC grown on collagen gel beads without migration seems to keep their original elongated morphology. (C-E) iMAECs collected after cell sorting were cultured for 24 h and imaged (C: phase contrast, D: DiI-Ac-LDL staining by fluorescence microscopy, and E: merged image). Scale bar = 50 μm.
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Figure 2: Morphology of mouse aortic endothelial cells. (A) Aortic explants were cultured on top of collagen gel beads for 4 days. EC grew and migrated out of the aorta piece. (B) EC grown on collagen gel beads without migration seems to keep their original elongated morphology. (C-E) iMAECs collected after cell sorting were cultured for 24 h and imaged (C: phase contrast, D: DiI-Ac-LDL staining by fluorescence microscopy, and E: merged image). Scale bar = 50 μm.

Mentions: The summary of the MAEC isolation and immortalization procedure is shown in Figure 1. During explant culture on collagen gel beads, MAEC migrated out of the aortic explants and gradually covered the gel within 3-4 days (Figure 2A, white arrows). When aortic explants were removed after 3-5 days of culture on the collagen bead, patches of endothelial cells were observed (Figure 2B, red arrows), suggesting that these are endothelial cells that did not migrate out (the red line indicates where the aortic explant was before it was removed) (Figure 2B). Both the migrating cells and the endothelial patches were cultured in a 24-well plate and immortalized by the polyoma middle T antigen method, which specifically immortalizes ECs [29,30]. Immortalized cells were positively selected via G418 antibiotic over a 4 to 6 week period. To further ensure endothelial purity, we used a stringent gating strategy to collect only a portion of immortalized cells that were singlets and showed strong DiI-Ac-LDL fluorescence signal using HUVEC and RASM as positive and negative controls, respectively. Figure 2C-E show typical morphology of iMAEC obtained after the flow cytometric sorting using DiI-Ac-LDL.


Development of immortalized mouse aortic endothelial cell lines.

Ni CW, Kumar S, Ankeny CJ, Jo H - (2014)

Morphology of mouse aortic endothelial cells. (A) Aortic explants were cultured on top of collagen gel beads for 4 days. EC grew and migrated out of the aorta piece. (B) EC grown on collagen gel beads without migration seems to keep their original elongated morphology. (C-E) iMAECs collected after cell sorting were cultured for 24 h and imaged (C: phase contrast, D: DiI-Ac-LDL staining by fluorescence microscopy, and E: merged image). Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230636&req=5

Figure 2: Morphology of mouse aortic endothelial cells. (A) Aortic explants were cultured on top of collagen gel beads for 4 days. EC grew and migrated out of the aorta piece. (B) EC grown on collagen gel beads without migration seems to keep their original elongated morphology. (C-E) iMAECs collected after cell sorting were cultured for 24 h and imaged (C: phase contrast, D: DiI-Ac-LDL staining by fluorescence microscopy, and E: merged image). Scale bar = 50 μm.
Mentions: The summary of the MAEC isolation and immortalization procedure is shown in Figure 1. During explant culture on collagen gel beads, MAEC migrated out of the aortic explants and gradually covered the gel within 3-4 days (Figure 2A, white arrows). When aortic explants were removed after 3-5 days of culture on the collagen bead, patches of endothelial cells were observed (Figure 2B, red arrows), suggesting that these are endothelial cells that did not migrate out (the red line indicates where the aortic explant was before it was removed) (Figure 2B). Both the migrating cells and the endothelial patches were cultured in a 24-well plate and immortalized by the polyoma middle T antigen method, which specifically immortalizes ECs [29,30]. Immortalized cells were positively selected via G418 antibiotic over a 4 to 6 week period. To further ensure endothelial purity, we used a stringent gating strategy to collect only a portion of immortalized cells that were singlets and showed strong DiI-Ac-LDL fluorescence signal using HUVEC and RASM as positive and negative controls, respectively. Figure 2C-E show typical morphology of iMAEC obtained after the flow cytometric sorting using DiI-Ac-LDL.

Bottom Line: Here, we developed an effective method to prepare immortalized MAEC (iMAEC) lines.Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen.Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wallace H, Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, 1760 Haygood Drive, Health Science Research Building, E-170, Atlanta, GA 30322, USA. hanjoong.jo@bme.gatech.edu.

ABSTRACT

Background: The understanding of endothelial cell biology has been facilitated by the availability of primary endothelial cell cultures from a variety of sites and species; however, the isolation and maintenance of primary mouse aortic endothelial cells (MAECs) remain a formidable challenge. Culturing MAECs is difficult as they are prone to phenotypic drift during culture. Therefore, there is a need to have a dependable in vitro culture system, wherein the primary endothelial cells retain their properties and phenotypes.

Methods: Here, we developed an effective method to prepare immortalized MAEC (iMAEC) lines. Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen. Immortalized cells were then incubated with DiI-acetylated-low density lipoprotein and sorted via flow cytometry to isolate iMAECs.

Results: iMAECs expressed common markers of endothelial cells, including PECAM1, eNOS, VE-cadherin, and von Willebrand Factor. iMAECs aligned in the direction of imposed laminar shear and retained the ability to form tubes. Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.

Conclusion: In summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.

No MeSH data available.


Related in: MedlinePlus