96 well angiogenesis μ plates

A tube formation assay was performed to assess the anti-angiogenic effect of bac I and II, either alone or in combination, on 2H-11 and HUVEC, as previously described [13 (link),58 (link)]. Briefly, 96-well angiogenesis μ-plates (Ibidi, Martinsried, Germany) were prepared by coating wells with Matrigel (Corning), according to the manufacturer’s instructions. In the respective complete medium, 2H-11 and HUVEC were resuspended, supplemented with either vehicle or bac I and/or II, and were plated at 1.5 × 10⁴ cells per well. After 2-h incubation, the number of loops formed was counted.

For the in vitro angiogenesis assay, spheroids were harvested and embedded into degradable 3.5 w/v% PEG hydrogels, functionalized with 0.5 mM linRGD and varying amounts of sHA-eSH (0–1 mg/mL). Hydrogels were prepared as described previously with 15 v/v% OptiPrep to prevent sedimentation of spheroids prior to polymerization of the hydrogel. For the polymerization step, the spheroid and crosslinker solutions were added to the functionalized PEG-VS and 10 μL hydrogels were formed in 96-well angiogenesis μ-plates (Ibidi GmbH, Germany). After polymerization, hydrogels were overlaid with 70 μL cell culture medium (basal medium with 15% FBS). For stimulation of the HUVECs, the cell culture medium was supplemented with 50 ng/mL VEGF₁₆₅. The gels were incubated at 37°C in a humidified 5% CO₂ atmosphere and imaged after 48 h. Phase-contrast images were acquired with a Zeiss Primovert (Zeiss, Germany), equipped with an Axiocam 208 color and 20 × objective (Zeiss, Germany, Plan-Achromat, Ph1, 20 ×/0.30). Sprouting was quantified by measuring the cumulative sprout length, which had grown out of each spheroid, using the imaging software FIJI.