Porcine aortic endothelial cells (PAEs) are isolated from the aorta of healthy pigs by collagenase digestion, and cultured on gelatin particles.
Cells are maintained at 37°C in a humidified 5% CO2/95% air incubator and cultured up to passage 6 in DMEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), glutamine (2.0 mmol/L), and calf serum (10%). Gelfoam is cut into 2.5×1.0×0.3-cm blocks. The Gelfoam blocks are hydrated in PBS and placed in 35×10-mm tissue culture dishes; 0.1 mL of a 0.8×106 cells/mL PAE cell suspension is placed directly on the sponge, thereby seeding 0.8×105 cells/block. The cells are placed at 37°C in a humidified 5% CO2/95% incubator and allowed to adhere for 2 hours. The cell-loaded Gelfoam blocks are subsequently placed in 17×100-mm polypropylene tubes containing 2 mL of medium and incubated for up to 2 weeks at a 45° angle. Growth medium is changed every 72 hours.
In some of the applications 4.0 × 1.0 × 0.3 cm3 blocks of sterile Gelfoam® were seeded with 1.25 –1.66 × 105cells/cm3 of Gelfoam® and incubated for ≈ 2 weeks until confluent in Endothelial Basal Medium supplemented with FBS (5%) and gentamicin (50 μg/mL).