Its media supplement

Primary adipose stem cells (ADSCs) were isolated from GDM mice and normal gestational mice, respectively. Under aseptic conditions, the abdominal cavity of the mice was opened, and AT from the abdominal and inguinal regions was obtained. The AT was then rinsed, minced, and collected in a pre-cooled Hank’s Balanced Salt Solution (Sangon BIOTECH, Shanghai, China). Next, 2 mg/ml collagenase I (Yeason, Shanghai, China) and 3 mM CaCl₂ were added in double volume to the tissue, which was then digested at 37 ℃ for 4 h. Digestion was stopped by adding an equal volume of DMEM/F12 medium (Gibco, GIBCO, NY, USA) containing 10% FBS (Merck KGaA, Darmstadt, Germany), followed by centrifugation at 1200 g for 10 min. The cell precipitates were resuspended, washed with PBS, and cultured in DMEM/F12 medium containing 10% FBS. Mouse normal liver cells, AML12, were purchased from Shanghai Fuheng Biotechnology and cultured in DMEM/F12 medium containing 10% FBS, 1% ITS media supplement (R&D Systems, MN, USA), and 40 ng/ml dexamethasone (Merck KGaA).

NCI-H295R (RRID: CVCL_0458) and SW-13 (RRID: CVCL_0542) were purchased from ATCC. CU-ACC1 (RRID: CVCL_RQ00) and CU-ACC2 (RRID: CVCL_RQ01) were provided by Dr. K. Kiseljak-Vassiliades (5 (link)). NCI-H295R cells were grown in 1:1 DMEM:F12 (Thermo Fisher Scientific) supplemented with 2.5% Nu-Serum (Corning), 1% ITS media supplement (R&D Systems), and 1% Penicillin–Streptomycin (Thermo Fisher Scientific). SW13 was grown in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (GeminiBio) and 1% Penicillin–Streptomycin (Gibco). CU-ACC1 and CU-ACC2 ACC cell lines were grown in F medium [3:1 (v/v) Ham's F-12 Nutrient Mixture–DMEM (Thermo Fisher Scientific) supplemented with 5% FBS, 0.4 µg/mL hydrocortisone (Sigma-Aldrich), 5 µg/mL insulin (Sigma-Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 10 ng/mL EGF (Invitrogen), 24 µg/mL adenine (Sigma-Aldrich)] (5 (link)). All cell lines were cultured at 37°C in a humidified incubator with 5% CO₂. Cell line identification tests were performed by short tandem repeat polymorphism analysis, using previously reported standard methods (5, 26 (link)). All cell lines were tested by MycoAlert Mycoplasma Detection Kit (Lonza) and confirmed to be Mycoplasma negative.