Angiogenic Potential of Mast Cells

To test the angiogenic potential of mast cells in the presence of healthy and degenerate IVD cells, media from the DCCM-Mast cell cultures was evaluated for blood vessel formation using the tubular assay. Human Umbilical Vein Endothelial Cells (HUVECs) were expanded in Medium 200 with Low Serum Growth Supplement (LSGS) (ThermoFisher) and seeded in 96 well plates at 4.0 × 10⁴ cell/well on wells pre-coated with 32 μl of Geltrex (ThermoFisher – A1413201)^{64 (link)}. Conditions included (1) Standard HUVEC media with LSGS (Positive control), (2) HUVEC media without LSGS (negative control), (3) media from mast cells in basal conditions, and (4) the healthy and (5) degenerate DCCM-mast cell groups (N = 4). Media from each group was filtered using PALL Microsep Advance Centrifugal Devices (PALL Corporation) to retain the soluble factors greater than 3,000 Da and resuspended in HUVEC media (Medium 200) without LSGS before being applied to HUVECs. HUVECs were incubated in conditions described above at 5%CO₂ at 37 °C for 6 hours. After incubation, 4 mM Calcein AM was added and images captured at ×4 magnification using TiE Nikon Inverted Microscope. The percent (%)area of tubular fluorescence was quantified using ImageJ⁸⁰ .