4.3. FaDu and Primary oral Squamous Carcinoma Cell Culture and Spheroid Formation

The FaDu human HNSCC cell line (ATCC Cat# HTB-43, RRID: CVCL_1218) was obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin solution at 37 °C in a 5% CO₂ humidified atmosphere. The cell line was tested for contamination every 2 months with the CellSafe Mycoplasma PCR detection kit (Cat# CS-D, CellSafe Co., Yongin city, Korea). In addition, to compare the anticancer effect of cisplatin on tumors in the nude mice, we established primary cells from HNSCC patient tumor tissue (T4N0M0, left buccal mucosa). Primary cells were obtained after 4 weeks in culture as adherent colonies in DMEM medium with 5% FBS. To obtain spheroids, cells after 3 passages were seeded into a 96-well U-bottom Ultra-Low Attachment plate (4000 cells/well) (Corning Inc., Tewksbury, MA, USA) and cultured for 2–3 days until spheroid formation (approximately 400 μm in diameter). When we analyzed the spheroid size with Cell³ iMager (Screen Holdings, Shiga, Japan), the diameter and surface volume of each spheroid were the same within the error range of 5%. After treatment with trypsin for the spheroid, the number of cells constituting the spheroid was counted.