Hanne Verswyvel

Chapter 3 │ Page 74 collected from the 96-well ULA plates, washed with PBS, and lysed following the manufacturer’s protocol. Intracellular ATP, serving as a marker of cell viability, was quantified through a firefly luciferase reaction, with the luminescence measured using the Spark® Cyto (Tecan). 2.5 Patient-Derived HNSCC Organoid Establishment and Experimental Processing 2.5.1. Legal considerations Organoids were derived from tissue resection fragments of HNSCC patients who underwent curative surgery in the course of their treatment at the Antwerp University Hospital. Patients approved the generation and experimental use of patient-derived HNSCC organoids via written informed consent. The study was approved by the UZA-UAntwerp Ethics Committee under study reference EC20/08/090. Tissue samples were collected via the Antwerp Biobank (Antwerp, Belgium; ID: BE71030031000). 2.5.2. Patient sample processing and organoid establishment HNSCC tissue resections were processed based on the protocol of Driehuis et.al.[38] In short, after collection, samples were stored in organoid medium supplemented with primocin at 4°C for up to 24 hours. For digestion, the tissue was finely minced on a cooled petri dish, and enzymatically dissociated in a 1:1 trypsin (0.25%) and culture medium solution for up to 60 minutes at 37°C. The resulting suspension was filtered (100 μm, 54200, Greiner), centrifuged (1000 rpm, 5 min, 4°C), and treated with RBC lysis bu er to remove red blood cells (5 min, RT). After washing, the pellet was resuspended in ice-cold Cultrex BME Type 2 (3533-010-02, Trevigen), and plated as 20 μL droplets onto a pre-heated 6-well plate. The plate was inverted to allow Cultrex solidification (30 min, 37°C), after which the domes were overlaid with fully supplemented HNSCC-PDO medium (Table 2). To support organoid formation, RHO kinase inhibitor Y27632 (10005583, Cayman Chemicals)

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