Limits...
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

Scheme of mouse aortic endothelial cell isolation and immortalization.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Scheme of mouse aortic endothelial cell isolation and immortalization.

Mentions: Initially, 4-week-old male mice were used for MAEC isolation. Each mouse was sacrificed by CO2 asphyxiation and cleaned using 70% ethanol. The abdominal and thoracic cavities were opened and the mouse was perfused, via the left ventricle, with 3-4 mL of sterile heparinized (10 U/mL) 1X Hank's buffered salt solution (HBSS, Cellgro). Most organs were removed except for the thoracic/abdominal aorta, which was left intact. Perivascular fat and adventitia were removed from the ventral side of the aorta. To decrease contamination of medial smooth muscle cells, the cleaned aortas were perfused with HBSS containing 0.5% Triton X-100 for 5 minutes. The aorta was then dissected out, rinsed 5 times with fresh HBSS, and placed in a sterile dish of cold HBSS. The aorta was then cut into small rings (~1 mm length) using a sterile scalpel. Then, each aortic ring was opened and was carefully laid on a collagen gel bead with the endothelial cells directly facing the gel. The collagen gel consisted of type I collagen (Bio-Rad) diluted with EGM2-MV (Lonza) to a final concentration of 1.75 mg/mL. Each collagen gel bead was prepared using 20 µl collagen and was allowed to solidify at 37°C for at least 30 min before use. Note that each aortic piece were positioned flat on the surface of the collagen gel. After tissue placement, the gel bead and aortic piece were kept hydrated with EGM2-MV and care was taken to avoid completely submerging and dislodging the aorta piece from the bead. The explants were cultured at 37°C and 5% CO2 in an incubator and monitored daily. The key steps of primary MAEC isolation are summarized in Figure 1.


Development of immortalized mouse aortic endothelial cell lines.

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

Scheme of mouse aortic endothelial cell isolation and immortalization.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Scheme of mouse aortic endothelial cell isolation and immortalization.
Mentions: Initially, 4-week-old male mice were used for MAEC isolation. Each mouse was sacrificed by CO2 asphyxiation and cleaned using 70% ethanol. The abdominal and thoracic cavities were opened and the mouse was perfused, via the left ventricle, with 3-4 mL of sterile heparinized (10 U/mL) 1X Hank's buffered salt solution (HBSS, Cellgro). Most organs were removed except for the thoracic/abdominal aorta, which was left intact. Perivascular fat and adventitia were removed from the ventral side of the aorta. To decrease contamination of medial smooth muscle cells, the cleaned aortas were perfused with HBSS containing 0.5% Triton X-100 for 5 minutes. The aorta was then dissected out, rinsed 5 times with fresh HBSS, and placed in a sterile dish of cold HBSS. The aorta was then cut into small rings (~1 mm length) using a sterile scalpel. Then, each aortic ring was opened and was carefully laid on a collagen gel bead with the endothelial cells directly facing the gel. The collagen gel consisted of type I collagen (Bio-Rad) diluted with EGM2-MV (Lonza) to a final concentration of 1.75 mg/mL. Each collagen gel bead was prepared using 20 µl collagen and was allowed to solidify at 37°C for at least 30 min before use. Note that each aortic piece were positioned flat on the surface of the collagen gel. After tissue placement, the gel bead and aortic piece were kept hydrated with EGM2-MV and care was taken to avoid completely submerging and dislodging the aorta piece from the bead. The explants were cultured at 37°C and 5% CO2 in an incubator and monitored daily. The key steps of primary MAEC isolation are summarized in Figure 1.

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