Coculturing Endothelial Cells and Fibroblasts for Vascular Network Formation

Human endothelial colony forming cell-derived endothelial cells (ECFC-ECs) were isolated from cord blood as previously described^{29 (link)} and expanded in gelatin-coated (0.5%) flasks in EGM-2 medium (Lonza). ECs were transduced with lentivirus constructs (Addgene) to express mCherry fluorescent protein and used once they reached passage 4–7. Normal human lung fibroblasts (NHLFs) were purchased from Lonza, cultured in DMEM (Corning) containing 10% fetal bovine serum (FBS, Gemini Bio Products), and used once they reached passage 4–7. All cell types were grown in a 37 °C/5% CO₂/20% O₂ incubator in 100% humidified air before use in experiments. Here, the fibroblasts served as pericytes in the coculture system. Fibroblasts can stabilize and promote the maturation of networks in microfluidic devices. The soluble protein secreted by fibroblasts supported not only EC sprouting but also lumen formation of the vascular networks. Fibroblast-derived proteins also increased the stiffness of the ECM, thus partially inducing lumen formation by ECs. Furthermore, fibroblasts can secrete a variety of angiogenic growth factors during tissue growth^{30 (link),31 (link)}.