Hi Paulo,

it should not be a big problem to grow HAEC. In my experience the EGM2 medium from Lonza is the best one for ECs, unfortunately also the most expensive one. Make sure that you coat your plates with gelatine or fibronectin, and split these cells quite moderate (1:2 or 1:3). Sk it should take them 2-3 days to reach confluency. It is normal that they change their morphology and look "swollen" after a couple of passages, that's why you want to use them at low passage numbers for your experiments.  

In my experience, you can passage the cells only 5-6 times from the original purchased primary cells (Lonza system).  Do not let the cells approach confluence before you passage them (~70-80% max) as confluent EC become quiescent and will not grow well even when diluted into new flasks. However, do not plate cells too sparsely either.  They like the "buddy" system—cells reasonably close by—to grow well.  I make liquid nitrogen freezer stocks of early passage cells (P3 and P4) and use P5 and P6 in experiments.  This way we can use the original batch of cells over a longer time period. When thawing cells, do not remove the DMSO cryopreservative by washing then plating cells. Plate cells first then next day change the medium to remove DMSO.  Make freezer stocks when cells reach 65-70% confluence—they must be actively growing or the viability upon thawing is poor. I use 40% FBS, 50% EGM-2 and 10% DMSO as freeze medium.  Freeze cells slowly.  After dispensing cells into cryo vials, I incubate them on ice for 30 min then put them in the slow freezing apparatus for my liquid N2 tank.  Thaw cells quickly.  Good Luck!

Hi, I use Promocell endothelial cell medium.  I have not found it necessary to coat the flasks for HAECs (although I do for more delicate microvascular ECs)...