Mesenchymal spheroids
Primary human fibroblasts (FMA73) were extracted from skin explants obtained through the elective breast surgery of a healthy young woman following informed consent; this tissue was provided by Walid Rachidi, CEA Grenoble. GFP- and RFP-labelled HUVEC cells (Angio-Proteomie, cat. no. CAP0001GFP and cat. no. CAP0001RFP, respectively) were cultured in complete EndoGM medium (Angio-Proteomie, cat. no. CAP02). Passage 5–7 cells were used for the experiments. Fibroblasts cultured in Fibroblast Growth Medium-2 (Lonza, cat. no. CC-3132), and passage 6–8 cells were used for the experiments. We prepared fibroblasts and HUVEC co-culture, termed mesenchymal spheroid model here, in U-shaped 96-well ultra-low attachment microplates (Corning, cat. no. CLS4515). Fibroblasts and HUVEC cells were mixed at a ratio of 1:1 (5000 cells per well) in 150?µl of medium consisting of a mix of CnT-ENDO (Cellntec, cat. no. CnT-ENDO) / CnT-Prime Fibroblast medium (Cellntec, cat. no. CnT-PR-F) at a ratio 1:1. After pre-culturing for 1 day in the microplate, a spheroid was introduced into the device. The same medium mix of CnT-ENDO / CnT-Prime Fibroblast medium was used for the microfluidic perfusion of the fibroblasts and HUVEC co-culture spheroids. RFP-HUVEC cells were suspended in the hydrogel at a concentration of 6×106 cells per ml.