| Nicosia and Ottinetti 1990 [41] | Present study 2017 |
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Application/novelty | • Developed a quantitative and reproducible angiogenesis assay for cell-free compounds • Tested the effect of inhibitory/stimulatory soluble factors on angiogenesis | • Modified a quantitative and reproducible angiogenesis assay for coculturing cells • A novel application to study the angiogenic function and potency of cell therapy candidates for clinical application and banking |
Set up | • Thoracic rat aorta embedded in fibrin or collagen • Prepared agarose wells to support aortic rings • Endothelial media: MCDB 131 to a mixture of 1:1 with DMEM F12 (FBS-free) • Stimulation of angiogenesis with mouse sarcoma 180 • Studied inhibition of angiogenesis using hydrocortisone | • Thoracic rat aorta embedded between two layers of Matrigel™ • Support of angiogenesis from EHS mouse sarcoma extract (Matrigel™) • Endothelial basal media supplemented with 2% FBS and 1% penicillin/streptomycin for cocultures • Prelabeled MSCs cocultured with developing endothelial networks for tracing |
Analysis | • Daily counting of newly developed microvessels (15 days) using bright-field microscopy • Florescent imaging of endothelial networks using FVIII-Ra immunohistochemistry | • Quantified radial network growth and network loops at day 5 following MSC coculture • Live-cell florescence imaging of MSC migration, integration site preference and morphology (coverage) in endothelial networks • Developed methods to extract MSCs and endothelial cells to perform downstream analysis including flow cytometry and qPCR |