Immortalized Stem Cells for Angiogenesis

hTERT immortalized human adipose-derived mesenchymal stem cells (hASCs/TERT1, Evercyte) were either used as purchased or retrovirally infected with green-fluorescent proteins (GFP-hASCs) according to the protocol by Knezevic et al. (2017)^{54 (link)}. Endothelial Cell Growth Medium-2 (EGM-2) was prepared by supplementing the EBM-2 Basal Medium with the EGM-2 SingleQuots Supplements from the EGM-2 Endothelial Cell Growth Medium-2 BulletKit (Lonza). hASCS/ GFP-hASCs were cultured in T-75 cell culture flasks (Greiner Bio-One) in EGM-2 supplemented with 10% fetal calf serum (FCS, Sigma-Aldrich). Human umbilical vein endothelial cells (HUVECs, PELOBiotech GmbH) were infected with red-fluorescent protein (RFP)-expressing lentiviral particles at passage 1. Zeocing resistant RFP-HUVECs were selected and cultured in EGM-2 supplemented with 5% FCS in T-25 cell culture flasks (Greiner Bio-One). All cell lines were cultivated in an incubator (BINDER) at 37 °C with 5% CO2 in a humid atmosphere and split at around 80 percent confluency. The media were changed every 2 to 3 days.