Gentamycin amphotericin b

HNmMM-derived primary cell lines were established from four fresh surgically excised HNmMM tissue samples from the original cohort of 20 patients by culturing them within a Matrigel explant prior to extraction of the cells following abundant growth in 24-well plates (Raylab, Auckland, New Zealand). The extracted cells were cultured in a cell culture medium consisting of DMEM (1X) and GlutaMAX-1 (cat#10569-010, Gibco, Rockford, IL, USA) supplemented with 10% fetal bovine serum (cat# 10091148, Gibco), 5% mTeSR 1 Complete medium (cat#85850, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin (cat#15140122, Gibco), and 0.2% gentamycin/amphotericin B (cat#R015-10, Gibco). All cultures were maintained in a humidified incubator at 37 °C with 5% CO₂. All primary cell lines used for the experiments were at passages 4–5 (RT-qPCR and WB) or 8–9 (tumorsphere formation).

mHNcSCC-derived primary cell lines were established from three freshly excised mHNcSCC tissue samples from the 15 patients included in the IHC staining, and cultured as explants by placing them between layers of Matrigel (cat#354234, Corning Life Sciences, Tewksbury, MA, USA) in 24-well plates and adding an explant culture media DMEM (cat#10569010, Gibco, Rockford, IL, USA) + 2% penicillin-streptomycin (cat#15140122, Gibco) + 0.2% gentamycin/amphotericin B (cat#R01510, Gibco). Cells were extracted from the Matrigel following abundant growth using Dispase (cat#354235, Corning Life Sciences). The extracted cells were cultured and passaged in a cell culture media consisting of DMEM (1X) (Gibco) and GlutaMAX-1 (cat#10569-010, Life Technologies), DMEM medium supplemented with 10% fetal bovine serum (cat#10091148, Gibco), 5% mTeSR™ (cat#85850, StemCell Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin (Gibco) and 0.2% gentamicin/amphotericin B (Gibco). All cultures were maintained in a humidified incubator at 37°C with 5% CO₂. All mHNcSCC-derived primary cell lines used for the experiments were at passages 4–8 (WB and RT-qPCR) or 7–9 (tumorsphere formation assays).

Primary cell lines were generated from four available freshly excised mHNcSCC tissue samples of the original cohort of 20 patients. Samples were cut into small pieces and incubated between layers of Matrigel (cat#354234, Corning Life Sciences, Tewksbury, USA) in 24-well plates with a culture media containing Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax™ (cat#15140122, Gibco, Rockford, IL, USA) supplemented with 2% penicillin-streptomycin (cat#15140122, Gibco) and 0.2% gentamycin/amphotericin B (cat#R01510, Gibco). Once sufficient cell growth was achieved to support transfer to a monolayer culture, cells were extracted by dissolving the Matrigel with Dispase (cat#354235, Corning Life Sciences) and transferred to an adherent culture flask with media consisting of DMEM (1X) (Gibco) with Glutamax™ supplemented with 10% fetal bovine serum (cat#10091148, Gibco), 5% mTeSR™1 Complete Medium (cat#85850, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin and 0.2% gentamycin-amphotericin (Gibco) in a humidified incubator at 37°C and 5% CO₂. Cells were expanded in culture and harvested between passages 4 and 8.