MDPI AG Acta Medica Okayama 2227-9059 12 10 2024 Development and Characterization of a Three-Dimensional Organotypic In Vitro Oral Cancer Model with Four Co-Cultured Cell Types, Including Patient-Derived Cancer-Associated Fibroblasts 2373 EN Yuka Aizawa Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Kenta Haga Division of Reconstructive Surgery for Oral and Maxillofacial Region, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Nagako Yoshiba Department of Oral Health and Welfare, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Witsanu Yortchan Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Sho Takada Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Rintaro Tanaka Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Eriko Naito Division of Oral and Maxillofacial Surgery, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Tatsuya Abé Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Satoshi Maruyama Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Manabu Yamazaki Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Jun-ichi Tanuma Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Kazuyo Igawa Neutron Therapy Research Center, Okayama University Kei Tomihara Division of Oral and Maxillofacial Surgery, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Shinsaku Togo Department of Respiratory Medicine, Graduate School of Medicine, Juntendo University Kenji Izumi Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Background/Objectives: Cancer organoids have emerged as a valuable tool of three-dimensional (3D) cell cultures to investigate tumor heterogeneity and predict tumor behavior and treatment response. We developed a 3D organotypic culture model of oral squamous cell carcinoma (OSCC) to recapitulate the tumor–stromal interface by co-culturing four cell types, including patient-derived cancer-associated fibroblasts (PD-CAFs). Methods: A stainless-steel ring was used twice to create the horizontal positioning of the cancer stroma (adjoining normal oral mucosa connective tissue) and the OSCC layer (surrounding normal oral mucosa epithelial layer). Combined with a structured bi-layered model of the epithelial component and the underlying stroma, this protocol enabled us to construct four distinct portions mimicking the oral cancer tissue arising in the oral mucosa. Results: In this model, ƒ¿-smooth muscle actin-positive PD-CAFs were localized in close proximity to the OSCC layer, suggesting a crosstalk between them. Furthermore, a linear laminin-ƒÁ2 expression was lacking at the interface between the OSCC layer and the underlying stromal layer, indicating the loss of the basement membrane-like structure. Conclusions: Since the specific 3D architecture and polarity mimicking oral cancer in vivo provides a more accurate milieu of the tumor microenvironment (TME), it could be crucial in elucidating oral cancer TME. No potential conflict of interest relevant to this article was reported.