Chick serum

Chicken lung explant culture was performed, as previously described [30 ], to study potential temporal metabolic changes while in controlled settings. Briefly, after dissection in PBS, lungs were placed on top of 8 µm nucleopore polycarbonate membranes (Whatman, USA) and incubated for 48 h in 200 µL of medium 199 (5.5 mM glucose; Sigma, USA) supplemented with 10% (V/V) chick serum (Invitrogen, USA), 5% (V/V) heat-inactivated fetal calf serum (Invitrogen), 1% (V/V) L-glutamine (Invitrogen), 1% (V/V) penicillin 5000 IU/mL plus streptomycin 5000 IU/mL (Invitrogen) and 0.25 mg/mL of ascorbic acid (Sigma). The medium was replaced by fresh supplemented medium at 24 h of culture. Lung explants were photographed at 0 h (D0), 24 h (D1), and 48 h (D2) with a camera (Olympus U-LH100HG) coupled to a stereomicroscope (Olympus SZX16). The medium was collected at D0, D1, and D2 for ¹H-NMR spectroscopy analysis. D0 and D2 lung explants were collected for RNA and protein extraction; D2 lung explants were collected for EdU proliferation assay and basal oxygen consumption rate assay.

Lungs were dissected in PBS and placed on 8 μm nucleopore polycarbonate membranes (Whatman, Marlborough, MA, USA). The lung explants were cultured in 200 μL of medium 199 containing 5.5 mM glucose (Sigma, St Louis, MI, USA),supplemented with 1% (v/v) L-glutamine (Invitrogen, Waltham, MA, USA), 0.25 mg/mL of ascorbic acid (Sigma), 5% (v/v) heat-inactivated fetal calf serum (Invitrogen), 10% (v/v) chick serum (Invitrogen), and 1% (v/v) penicillin 5000 IU/mL plus streptomycin 5000 IU/mL (Invitrogen). The lung explants were exposed to increasing doses of BMS (BMS493, Sigma): 0.1 μM, 1 μM, or 10 μM; or to a different experimental setting with 1 μM of RA (Sigma) or 10 μM of BMS. DMSO 0.1% was used as the control. The lung explants were incubated for 48 h at 37 °C with 5% CO₂ (Heraeus HeraCell CO₂ incubator, Hanau, Germany). At 24 h, the culture medium was replaced by a fresh supplemented medium. The lung explants were photographed at 0 h (D0), 24 h (D1), and 48 h (D2) (Olympus U-LH100HG coupled to Olympus SZX16) and then morphometrically analyzed (AxioVision, Carl Zeiss Microscopy, Oberkochen, Germany). Media samples were collected at D0, D1, and D2 for ¹H-NMR spectroscopy. D2 tissues were collected for RNA, DNA, and protein extraction. D2 explants were also collected for in situ hybridization, proliferation assay, and seahorse analysis.

Collagen gels at a concentration of 2 mg/mL collagen were made using 3 x Dulbecco’s Modified Eagle’s Medium (Life Technologies), 10% chick serum (Life Technologies), sterile 18 MΩ water, 0.1 M NaOH, and rat tail collagen I (BD Biosciences). An aliquot of the collagen gel solution was pipetted into the wells in the silicone sheet and allowed to solidify for 1 hr at 37 °C and 5% CO₂. The dissected outflow tracts were then placed on top of the collagen gel, and excess media was pipetted off to allow for the valve primordia to come in contact with the collagen gel. After 6 hr of incubation at 37 °C and 5% CO₂. the valve endocardial cells are repolarized, delaminated, and attached to the surface of the collagen constructs. These endocardial cells were then exposed to USS or OSS at 2 or 20 dyne/cm² for 24 hr.