Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

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.

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. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's 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.
3. 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 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes. Discard supernatant.
6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 2 X 103 to 3 X 103 viable cells/sq. cm is recommended.
7. Incubate cultures at 37°C. Subculture when cell concentration reaches between 8 X 104 and 1 X 105 cells/sq. cm.

Interval: Twice a week

Subcultivation Ratio: A seeding density of 2 X 103 to 3 X 103viable cells/sq. cm should be used when subculturing these cells

Medium Renewal: Every 2 to 3 days

Freeze medium: Complete Growth medium, 95%; DMSO, 5%

Storage temperature: liquid nitrogen vapor phase

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37°C