Hacat

Human oral tongue cancer cell lines, HSC-3 (JCRB Cat# JCRB0623, RRID:CVCL_1288, passages 4–12) and OSC-20 (Cat# JCRB0197, RRID:CVCL_3087, passages 4–12) were obtained from the Japanese Collection of Research Bioresources Cell Bank. The spontaneously immortalized human keratinocyte cell line HaCaT (RRID:CVCL_0038, passages 4–12) was purchased from AddexBio Technologies (Cat# T0020001, San Diego, CA). Human dysplastic keratinocyte cell line DOK (RRID:CVCL, 1180, passages 4–12) was purchased from Sigma-Aldrich (Cat# 94122104). The cell lines harbor mutations in TP53. Further information on the mutational status of the HSC-3, OSC-20 and DOK cell lines can be obtained from COSMIC, the Catalogue Of Somatic Mutations In Cancer (https://cancer.sanger.ac.uk).^{[98 (link)]} The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA, HSC-3, DOK, HaCaT) or DMEM/F12 (OSC-20) supplemented with fetal bovine serum (FBS) and penicillin/streptomycin (50 U/mL) for 48 hours. Cell lines were cultured in 75 cm² cell culture flasks at 37°C with 5% CO₂, 21% O₂ (normoxia). When cells reached 70–80% confluency, the culture medium was replaced with media supplemented with 10% exosome-depleted FBS (Gibco) and cultured for a further 48 hours for preparation of exosomes. Cell culture supernatant was collected and used for isolation of exosomes.