Dmem nutrient mixture f12 ham medium

Isolated CAFs and NOFs from OSCC patients and healthy donors were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; D6429, SIGMA) supplemented with 10% heat-inactivated newborn calf serum (NBCS; 31765068, GIBCO). OSCC cell lines UK1 (29 (link)) and Luc4 (30 (link)) were grown in DMEM/Nutrient Mixture F-12 Ham medium (D8437, Sigma) supplemented with 10% NBCS, 1× Insulin-Transferrin-Selenium (41400-04, Thermofisher Scientific), 0.4 μg/ml hydrocortisone (H0888, Sigma), 50 μg/ml L-ascorbic acid (A7631, Sigma), and 10 ng/ml epidermal growth factor (E9644, Sigma). All cell lines were propagated in humidity incubator at 5% CO₂ and 37°C temperature and regularly tested for mycoplasma contamination.

Primary chondrocytes were harvested from the epiphyseal cartilage of the knee joints and femoral heads of newborn C57BL/6 mice, as reported previously (Gosset et al., 2008 (link)). Briefly, cartilage was digested with 3 mg/mL collagenase D (Roche) in culture medium, which was a mixture of Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Sigma-Aldrich, St. Louis, MO, United States) and DMEM/nutrient mixture F-12 Ham medium (Sigma-Aldrich) supplemented with 5% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). Cells (5 × 10⁵) were seeded in a 10-cm dish and cultured in a culture medium without 1% penicillin/streptomycin. After the cells were confluent, they were passaged into 24-well plates and cultured for 3–5 days. Then, 100 or 330 ng/mL recombinant SHH (rSHH) (Peprotech, Rocky Hill, NJ, United States) was added, and the cultures were incubated for 24 h under hypoxia (1% O₂) using the BIONX-1 hypoxic culture kit (Sugiyamagen, Tokyo, Japan).