4.8. 3D Collagen Co-Culture Assay

Lentivirus green fluorescent protein transduced HUVECs (HUVECs-GFP) and lentivirus discosoma sp. Red fluorescent protein transduced pericytes (pericytes-DsRED) were mixed at 5:1 ratio in co-culture medium, which is basal EBM medium supplied with 2% FCS, rhFGF-B, ascorbic acid and 100 UmL^-1 PS. Cell mixture supplied with growth factors, including IL-3, SCF-1 and CXCL12 (BD Bioscience), at the volume of 300 µL was added to 200 µL bovine collagen type I (Gibco). NaOH was used to adjust pH to 7.5. Cell-collagen mixture was added to 96-well plate (50 µL per well). After 1 h incubation, 100 µL EGM2 was added per well and the plate was incubated overnight. The following day IS or control buffer was added to the cells. Images were taken from day 1 till day 3 after stimulation using inverted fluorescence microscope and analysed by AngioSys 2.0. To correct for batch effects, an additional condition of cells cultured in the standard co-culture medium was included in each independent experiment.