A little backstory on the protocol and experimental methods:

Our goal is to isolate primary embryonic coronary endothelial cells. At embryonic day e14.5, we harvest pregnant mice. The embryonic hearts are removed and placed in cold, sterile 1X PBS. From here, we utilize the Miltenyi Biotec Neonatal heart dissociation kit with the GentleMacs machine. Following heart homogenization, we use Miltenyi Biotec Neonatal Endothelial Cell Isolation Kit with both LS and MS columns. Following isolation, the cells are spun down in EGM2-Mv (Lonza EBM2 w/ bullet kit). They are then plated onto sterile 24 well plates containing cell culture coverglass submerged in Fibronectin (Corning) (2 hour incubation time before removal).

Typically, we wait around 24-48 hours to begin our treatments. Recently, we have attempted to wait around 4-6 days for the cells to reach confluency before adding our treatments. Our most recent endothelial cell isolation resulted in around 550,000 endothelial cells. 12,000 cells were seeded into each well and EGM2-Mv was added as a growth medium. The cells have been rinsed every 2 days with sterile 1X PBS and the EGM2-Mv has been replaced. Most cells seem to die off within the first 24 hours leaving us with significantly less cells in each well (most probably 5,000 cells remaining).

The cells seem to not grow regardless of the growth medium used. We initially quantified our treatment groups and noticed no significant change from control, even when one treatment was 100 ng murine VEGFA. We have resulted to explant cultures at e10.5 which have been working great but our goal is to successfully isolated coronary endothelial cells.

Our next idea is to not isolate the endothelial cells from the cardiomyocytes and other cells, therefore we will skip using the MS column from the Miltenyi protocol. We don't know if the endothelial cells require other cell species in order to grow or not.

If anyone has any recommendations for refining our protocol, any suggestions would be greatly appreciated.