Isolation of Primary Head and Neck Cells

Patient samples (n=4) were obtained from the Loma Linda University School of Medicine, Department of Otolaryngology and Head/Neck Surgery through the Loma Linda University Tissue Biorepository. Patients were all males with ages ranging from 65 to 77 years and the tissues were obtained following laryngectomy and glossectomy. The freshly resected tissues were kept on ice until ready for processing. Samples were transferred to petri dishes and washed with PBS-2X Gentamicin 3 times. Samples were minced using a sterile razor blade until they appeared as puree. The puree was passed over a 70 µm strainer using a plunger from a 3 mL syringe. Plain DMEM was used to wash the cells. The cells were then centrifuged at 1500 rpm for 10 mins. If pellet was red, red blood cells were removed using Ficoll. Otherwise, cells were counted using an automated cell counter and resuspended in three parts Ham’s F12, one part DMEM (Fisher Scientific) supplemented with 5% FBS (Omega Scientific), 10uM insulin, 0.4uM hydrocortisone, 2ug/ml isoprenaline, 24ug/ml adenine (chemicals from Sigma-Aldrich), 100U/ml penicillin, 10ug/ml streptomycin (Fisher Scientific). 5-10 uM Y27632 (BioGems) was added to establish growth in vitro (26 (link)). Cells were plated at 2 X 10⁶ in 6 well plates. Cells were monitored over time for development of clones that were then used to establish primary cultures.