Angiogenesis Assay for Primary Endothelial Cells

Tube formation assay was used as an in vitro angiogenesis model to validate 1°CEC function as previously described [36 (link)]. Briefly, Geltrex™ (Life Technologies, Grand Island, NY, USA) was thawed overnight at 4 °C. Using cold pipette tips, 10 μL/well of Geltrex™ was added to a μ-slide angiogenesis plate (ibidi GmBH, Gräfelfing, Germany). The Geltrex™ solidified into a thin layer after incubation at 37 °C for 1 h. 1°CECs were plated at a density of 12,000 cells/well and incubated at 37 °C, in 5% CO₂. After 3 h, network formation was imaged using an inverted phase-contrast microscope (Zeiss, Axio Observerer.D1, Whiteplains, NY, USA).