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Restrictions:
For-profit institutions must obtain license from University of North Carolina, Chapel Hill.
Antigen Expression:
Factor VIII-related antigen; Homo sapiens, expressed
The human umbilical vein cell line, EA.hy926, was established by fusing primary human umbilical vein cells with a thioguanine-resistant clone of A549 by exposure to polyethylene glycol （PEG）. Hybrid clones were selected in HAT medium and screened for factor VIII-related antigen. EA.hy926 cells have been maintained for more than 100 population doublings （PDLs）. Electron photomicrographs demonstrate cytoplasmic distribution of Weibel-Palade bodies and tissue-specific organelles, characteristics of differentiated endothelial cell functions such as angiogenesis, homeostasis/thrombosis, blood pressure and inflammation. [PubMed: 6407019] [PubMed: 2079463]
ATCC complete growth medium: The base medium for this cell line is ATCC-formulated Dulbeccos Modified Eagles Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10. Atmosphere: air, 95; carbon dioxide （CO2）, 5 Temperature: 37.0°C
Subculturing:
Protocol: Volumes used in this protocol are for 75 sq cm flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Remove and discard culture medium.
Briefly rinse the cell layer with Ca++/Mg++ free Dulbeccos phosphate-buffered saline （D-PBS） or 0.25 （w/v） Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed （usually within 5 to 15 minutes）. Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Transfer cell suspension to a centrifuge tube and spin at approximay 125 X g for 5 to 10 minutes. Discard supernatant.
Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 2 X 10（3） to 3 X 10（3） viable cells/sq. cm is recommended.
Incubate cultures at 37C. Subculture when cell concentration reaches between 8 X 10（4） and 1 X 10（5） cells/sq. cm. Interval: Twice a week Subc*tion Ratio: A subc*tion ratio of 1:6 to 1:10 is recommended Medium Renewal: Every 2 to 3 days
Recommended medium （without the additional supplements or serum described under ATCC Medium）:ATCC 30-2002 recommended serum:ATCC 30-2020 0.25 （w/v） Trypsin - 0.53 mM EDTA in Hank BSS （w/o Ca++, Mg++）:ATCC 30-2101 phosphate-buffered saline:ATCC 30-2200 Cell culture tested DMSO:ATCC 4-X Erythrosin B vital stain solution:ATCC 30-2404 Trypan Blue vital stain solution:ATCC 30-2402
References:
92902: Edgell CJ, et al. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl. Acad. Sci. USA 80: 3734-3737, 1983. PubMed: 6407019 92903: Bauer J, et al. In vitro model of angiogenesis using a human endothelium-derived permanent cell line: contributions of induced gene expression, G-proteins, and integrins. J. Cell. Physiol. 153: 437-449, 1992. PubMed: 1280276 92904: Edgell CJ, et al. Endothelium specific Weibel-Palade bodies in a continuous human cell line, EA.hy926. In Vitro Cell. Dev. Biol. 26: 1167-1172, 1990. PubMed: 2079463 92905: Rieber AJ, et al. Extent of differentiated gene expression in the human endothelium-derived EA.hy926 cell line. Thromb. Haemostasis 69: 476-480, 1993. PubMed: 8322270
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