Plate angiogenesis 96 well

The medium used in all organotypic 3D cultures was DMEM/F12 supplemented with 15% iFBS, 5% l-glutamine, 2.5% Pen-Strep, 1 μg/ml hydrocortisone, 0.2 U/ml insulin, 0.1 nmol/l cholera toxin and 25 ng/ml EGF (modified from [22 (link)]). In indicated experiments, the medium was further supplemented with Slit2 recombinant protein (0.5 µg/ml).
For imaging purposes and to perform cell viability assay, organotypic 3D cell culturing was performed primarily as described previously [23 (link)]. Briefly, cells were seeded as single cells between growth factor reduced Matrigel layers (Corning #356231) on 96-well angiogenesis µ-plates (Ibidi GmbH) in the density of 2000 cells/well. After Matrigel polymerization, medium was gently added on the top and replaced with fresh medium every 2–3 days. The formation of organoid-like structures was followed up to 12 days.
To extract RNA and protein lysates from organotypic 3D cell cultures, the cells were suspended in Matrigel to final concentration of 4 mg/ml and in the density of 250,000 cells/ml, and seeded on pre-heated tissue culture plates as drops (vol 80 µl). The dishes were first kept up-side down for 30 min in 37 °C until Matrigel was solidified. Subsequently, the medium was gently added to cover the drops and it was changed every 2–3 days during the 12 days culturing period.

Briefly, 1·10⁴ DiR-labeled or untreated ECFCs (n:4) were seeded, in triplicates, into a µ-plate angiogenesis 96 well (ibidi, 89,646) pre-coated with 10 µl Matrigel (BD Bioscience, 256,231), as described [39 (link)]. Images were registered after 24 h, 48 h and 72 h, with a Moticam 3.0 camera connected to an inverted phase-contrast microscope, under 4X magnification. The number of meshes and total length of segments in each well were quantified using the Angiogenesis Analyzer plugin with Image J v.2.0.

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.

The morphogenic potential of HUVECs and HMVEC-dLyAd cells to form capillary-like structures in vitro was evaluated by seeding cells on Matrigel, a basement membrane matrix (Corning, NY, USA). 10 μL of Matrigel was dispensed into each well in a µ-Plate Angiogenesis 96-well (ibidi GmbH Martinsried, Germany) and allowed to polymerize for 1 h at RT. HMVEC-dLyAd cells and HUVECs (12,000 cells/well) were synchronized by starvation in a serum-free medium for 3 h prior to seeding on the polymerized gel. 1.5 h after seeding, TNP-470 in various concentrations (0–50 μM) was added and left to incubate overnight. Images were taken using an inverted fluorescence microscope (Nikon ECLIPSE Ni-E). The mean vessel length and the total number of end points were quantified using the AngioTool image analysis software, n = 8.