Fabrication of a Three-Dimensional Spheroid Culture System for Oral Squamous Cell Carcinomas Using a Microfabricated Device

Simple Summary Cancer stem cells (CSCs) retain their ability to self-renew and differentiate and exhibit resistance to chemotherapy and radiotherapy. Therefore, the selective eradication of CSCs is the most rational method of cancer treatment. However, the presence of CSCs in cancer tissues and cell lines is extremely low, making it difficult to isolate and collect sufficient quantities of CSCs for further studies. We used microfabrication technology to develop a device that can easily generate uniform oral cancer cell spheroids in large quantities. The spheroids produced in the microwell showed an increased expression of CSC markers and resistance to anticancer drugs, suggesting that our device could be useful for high-throughput studies on oral CSCs.Abstract Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.