Angiogenic Potential of Cell Secretomes

Tube formation assay was performed as described previously (26 (link)). Briefly, 10 µL Matrigel Matrix GFR (Corning, Fisher Scientific, Schwerte, Germany) was added into a µ-plate angiogenesis 96-well (Ibidi, Gräfelfing, Germany). Human umbilical vein endothelial cells (HUVECs, PromoCell, Heidelberg, Germany) in passage two to five were grown to 80% confluence in Endothelial Cell Growth Medium (ECGM, PromoCell, Heidelberg, Germany), trypsinized, and cell pellet was resuspended in either PtF/CAF/hOFs supernatants from patients #3, #4 and #8, fresh supplemented ECGM, or HUVEC-conditioned FGM supernatant. The cells were then plated into the µ-plate 96-well at a density of 1x10⁴ cells/well and cultured for eight hours. Photographs of each well were taken with a Leica DMi8 microscope at five-fold magnification in phase contrast. At least five technical replicates of each sample were taken, the result of three independent experiments are displayed. The images were analyzed using ImageJ’s “Angiogenesis Analyzer” module. Tubes were determined and quantified as a measure of angiogenic potential.