Human umbilical vein endothelial cells (HUVEC) are isolated from the umbilical cord. The cells grow in confluent monolayers with a cobblestone-like morphology. The cells are homogenous, closely opposed, large, and polygonal with an oval, centrally located nucleus and indistinct cell borders. In any given section of suspended endothelial cells, approximately 30% contain Weibel-Palade bodies.
The umbilical cord is severed from the placenta soon after birth, placed in a sterile container filled with cord buffer (NaCl (0.14M), KCl (0.004M), phosphate buffer pH 7.4 (0.001M), glucose (0.011M)) and stored at 40C until processing. Cords should be discarded if stored for more than 3 h. The cord is inspected, and all areas with clamp marks are cut off. The umbilical vein is cannulated with a blunt 14-gauge, 2-cm long, needle; the needle is secured by clamping the cord over the needle with an umbilical cord tie. The vein is perfused with 100 ml of cord buffer and allowed to drain. The other end of the umbilical vein is then cannulated with a blunt, hubless, 12-gauge needle shaft with polyethylene tubing. 10 ml of collagenase (0.2%) in cord buffer is then infused into the umbilical vein, and the polyethylene tubing is clamped shut with a hemostat. The umbilical cord, suspended by its ends, is placed in a water bath containing cord buffer and incubated at 370C for 15 min. After incubation, the collagenase solution containing the endothelial cells is flushed from the cord by perfusion with 30 ml of cord buffer. The effluent is collected in a sterile 50 ml conical centrifuge tube containing 10 ml of Medium 199 (TC 199) with fetal calf serum (20% FCS). The cells are sedimented at 250 g for 10 min and washed once with 20 ml of TC 199-FCS (20%). The cells are then resuspended in 5 ml of fresh culture medium. The yield from this procedure is in the range of 0.5-1.5 X 108 cells. The cell suspension is divided equally among 4-6 plastic 35-mm Petri dishes. Sufficient medium is then added to make a final volume of 2 ml/dish. Endothelial cells are cultured in TC 199 containing FCS (20%), penicillin (200 U/ml), streptomycin (200µg/ml), and L-glutamine (2 mM). The dishes are incubated at 370C under 5% C02. The cells are fed twice a week with a complete change of fresh culture medium. For subculture, cells are harvested with EDTA(0.01%)-collagenase (0.1%) 1:4 - 1:8, every 10-14 days.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
