Trypsin edta solution 10

Commercially available, cryopreserved human umbilical vein endothelial cells (HUVEC), from pooled donors were acquired from Lonza (C2519A) and cultured in endothelial cell growth medium (EGM-2 BulletKit, CC-3162, Lonza) with 1% (v/v) gentamicin (10 ng mL -1 , Gibco). HUVECs at passage three were thawed and seeded in 175 cm 2 filter cap cell culture flasks (CELLSTAR, Greiner Bio-One). After 24 h, the cell culture media was exchanged and the HUVECs were expanded for the following 3 days before cell loading into the disc.
For generating a cell suspension for subsequent cell loading, adherent cells were washed with PBS (Dulbecco's phosphate buffered saline w/o calcium w/o magnesium, Biowest), detached by a 3 min incubation step at 37 °C using 0.05% (v/v) trypsin (Trypsin-EDTA Solution 10×, SIGMA Life Science) in Versene solution (Versene 1 : 5000 1×, Gibco). The cell suspension was transferred into a centrifuge tube (50 mL CELLSTAR polypropylene tube, Greiner Bio-One), trypsin inactivated by adding 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific) and centrifuged for 5 min at 1000 rpm or 216g (Multifuge 3S-R, Heraeus). Cells were counted using trypan blue (trypan blue 4 g l -1 in aqueous solution, VWR chemicals) using a hemocytometer (C-Chip Neubauer improved DHC-N01, NanoEnTek).