Methylcellulose

Spheroids were prepared as previously described [28 (link)]. Briefly, transfected BAECs were cultured in DMEM containing 0.3% methylcellulose (Sigma-Aldrich), 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin in U-shaped 96-well plates for 24 h to allow spheroid formation. Spheroids were transferred in complete medium containing 50% collagen (Corning, Corning, NY, USA) pH 7.4, and 0.6% methylcellulose and cultured for 24 h before fixation in 4% PFA. Image acquisitions were performed using an inverted Axio-observer Z1 epifluorescent microscope (Zeiss, Germany) with a 20 × objective. The numbers of sprouts and filopodia lengths were determined manually using ZEN Blue 2.3 (Zeiss). Filopodia were defined as actin-rich extensions from the tip-cells of the sprout with < 0.1 μm diameter. For length quantification, only filopodia visible in one focus plane were manually counted by drawing lines from the bases of the cells to the tips of the filopodia using the line tool of ZEN Blue.

PECs were harvested by peritoneal lavage with 10 ml of complete D5 ((DMEM (Corning), 5% FBS (Biowest), 2mM L-glutamine (Gibco), 1% HEPES (Corning), 10 U/mL Penicillin and 10 μg/mL streptomycin sulfate (Corning)) and washed once. The PECs were resuspended in 1 ml of D5, then were homogenized with 1 mm zirconia beads (BioSpec Products) in a Precellys 24 (Bertin Instruments) at 5000 rpm for 1 min. 10-fold serial dilutions were plated on 3T12s. Virus was absorbed for 1 hour at 37 degrees C before 1% (w/v) methylcellulose (Sigma-Aldrich) (10 g methylcellulose in 1L MEM (Corning)) was added as an overlay. Plates were incubated at 37 degrees C in 5% CO₂ for 7 days before fixing with 2% formaldehyde and staining with 0.1% crystal violet (Sigma-Aldrich).

HUVECs were trypsinised and resuspended in ECM culture medium containing 0,6 gr/L methylcellulose (Sigma Aldrich). Cells were seeded (400 cells per well in 100 µl) in U-bottom 96 well plates (Costar) and cultured for 24 h at 37 °C and 5% CO₂ to allow for formation of spheroids. The spheroids were collected and resuspended in FBS (Sciencell) containing 2,4 gr/L methylcellulose and mixed 1:1 with collagen I solution containing 3,77 g/L collagen I (Corning, USA), 10% M199 medium (Sigma Aldrich), 0,018 M HEPES and 0.2 M NaOH to adjust pH to 7,4. The mixture with the spheroids was allowed to polymerize for 30 min in a 24 well plate. To induce sprouting, 100 µl of ECM with or without VEGF (50 ng/mL, Preprotech) was added to the gels. Spheroids were fixed after 24 h using 10% fomaldehyde in PBS and visualized using an Olympus IX50 microscope. The cumulative sprout length per spheroid was measured using ImageJ software.