Fibronectin coated tissue culture plates

CFU-ECs were produced according to the protocol initially described by Hill et al. [25 (link)] and adapted by Smadja et al. [43 (link)]. Mononuclear cells (MNCs) were obtained by density gradient isolation and cultured with the EndoCult® Liquid Medium Kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. In short, MNCs were re-suspended in complete EndoCult® medium and seeded at 5 × 10⁶ cells/well in fibronectin-coated tissue culture plates (BD Biosciences, San Jose, CA, USA). After 48 h, to obtain CFU-ECs, non-adherent cells were collected and plated in EndoCult® buffer at 10⁶ cells/well in 24-well fibronectin-coated plates. CFU-EC colonies were counted after another 3 days, as recommended by the manufacturer.

Clonogenic assays allowing the enumeration of Colony Forming Unit- endothelial cells (CFU-EC) after ex vivo culture of peripheral blood mononuclear cells have been performed according to the method described previously by Hill et al. [4 ], and adapted by Smadja et al. [59 (link)]. PBMC were obtained by density gradient isolation and cultured with the Endocult^® Liquid Medium Kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. Briefly, PBMC were re-suspended in complete Endocult^® medium and seeded at 5 × 10⁶ cells/well in fibronectin-coated tissue culture plates (BD Biosciences, San Jose, CA, USA). After 48 h, to obtain CFU-ECs, non-adherent cells were collected and plated in Endocult^® medium at 10⁶ cells/well in 24-well fibronectin-coated plates. CFU-EC colonies were counted after another 3 days, as recommended by the manufacturer. This assessment was performed in 220 subjects.