Fibroblast growth factor 2 fgf2

hSMG tissues were collected after surgery and stored in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) after surgery. To culture primary cells from hSMGs, the tissues were transferred to a biosafety cabinet, cut into small pieces, and placed at the bottom of culture bottles (Corning). After incubation at 37 °C in 5% CO₂ for 4 h, epithelial cells were cultured in keratinocyte medium (KM; Sciencell) with supplements (KM-S) containing 5 μg/mL bovine serum albumin (BSA; Sigma-Aldrich), 5 ng/mL fibroblast growth factor-2 (FGF2; Sigma-Aldrich), 1 ng/mL epidermal growth factor (EGF; Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 5 μg/mL transferrin (Sigma-Aldrich), and 0.5 μg/mL hydrocortisone (Sigma-Aldrich); mesenchymal cells were cultured in DMEM-S, DMEM containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen). The medium was changed every 3 days. Primary cells were digested with 0.25% trypsin (Invitrogen), harvested by centrifugation, resuspended in medium, and reseeded in a cell-culture dish as passage 1 (P1). The digestion time was 4 min for epithelial cells and 1.5 min for mesenchymal cells. Cells at 80–90% confluence were passaged at a 1:3 split ratio, and the medium was changed every 2–3 days.

mSGOs are structures derived from SGOs, containing differentiated cells of the SG. In order to make mSGOs, SGOs were first generated in self-renewal conditions, either in control SGPC medium or with additional 1 μM Iso. At the end of the passage, gels were dissociated using Dispase, collected by centrifugation, and re-suspended in 25 μL of SG medium. This suspension was then mixed with 50 μL of growth-factor-reduced Matrigel and deposited as a drop in the center of a 12-well plate. After incubation for 20 min at 37° to solidify, 1 mL of enhanced SG medium was added, with 1 μM Iso, or 1 μM Met, as required. Enhanced SG medium was composed of 20 ng/mL epidermal growth factor (EGF) (Sigma-Aldrich), 20 ng/mL fibroblast growth factor 2 (FGF2) (Sigma-Aldrich), N2 (Invitrogen), 10 mg/mL insulin (Sigma-Aldrich), 1 mM dexamethasone (Sigma-Aldrich), and 10 μM Rho Kinase Inhibitor (Abcam). Cultures were maintained for 14 days, and medium was supplemented at day 3 and day 6 (0.5 mL of medium volume added). mSGO formation efficiency was calculated by quantifying the number of structures with at least three branches terminating in round end buds. mSGOs were enumerated as a proportion of seeded number of SGOs.

The co-culture was established as described earlier (Sarkanen et al. 2011 (link)). Briefly, fibroblasts (p 6–7), were seeded at 20,000 cells/cm² in fibroblast medium (Table 1) in 48-well plates for immunocytochemical and qRT-PCR analyses and in MEA-platforms and 12 mm diameter cover slips for functional analyses and grown for 2–3 days to confluency. HUVEC were seeded on top of confluent fibroblast cultures at 4000 cells/cm² in EGM-2 medium (Table 1). The day after cell seeding, angiogenic stimulation medium containing EBM-2 (Lonza), 2% FBS, 1 mM l-glutamine, 10 ng/ml vascular endothelial growth factor (VEGF, Sigma) and 1 ng/ml fibroblast growth factor 2 (FGF-2, Sigma) (Table 1) was applied to cells. The angiogenic stimulation medium was changed twice during the 6 day co-culture prior to CM seeding.