Hanne Verswyvel

Chapter 3 │ Page 72 Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies), supplemented with fetal bovine serum (FBS, 10%, Life Technologies), L-glutamine (1%, Life Technologies), and Penicillin/Streptomycin (P/S, 1%, Life Technologies). Cell cultures were maintained in a humidified atmosphere at 37°C and 5% CO2 conditions, and passaged upon reaching 80% confluence to ensure exponential growth. Cell identity was verified through short tandem repeat profiling, and routine mycoplasma contamination checks were performed. 2.2. HNSCC Spheroid Generation Three-dimensional (3D) tumor spheroids were generated for all included human HNSCC cell lines. Optimal cell seeding concentration depended on the cell line; with 4×10⁴ cells/mL for Cal27 and SCC22B, and 5×10⁴ cells/mL for SCC61 (100 μL/well). Cells were seeded into round-bottom, ultra-low attachment (ULA) 96-well plates (7007, Corning®) to stimulate condense, spheric formation while preventing surface attachment of the cells. The cell suspension was supplemented with 2% Matrigel DB Phenol Red Free (354230, Corning®) to further support spheroid condensation. After three days of undisturbed incubation, spheroids measuring 350-450 μm in diameter were achieved and used for further experiments. 2.3. NTP-Cisplatin Combination Treatment Application 2.3.1. NTP treatment A microsecond-pulsed dielectric barrier discharge (DBD) system, previously described in our work[25, 34-37], was used for this research. The set-up comprised a microsecond-pulsed power supply (Megaimpulse Ltd, Russia), with a roundbottom dielectrically covered electrode, ensuring an electrically and thermally stable plasma[25]. Operation parameters are detailed in Table 1.

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