Ex vivo culture of tumor cells from N-methyl-N-nitrosourea–induced bladder cancer in rats: Development of organoids and an immortalized cell line

Objective: We ex vivo cultured primary tumor cells from N-methyl-N-nitrosourea (MNU)-induced bladder tumors in rats and established an immortalized cell line from them. Materials and methods: Bladder tumors in rats were induced by instillation of MNU into the murine bladder. Primary tumor cells were prepared by the cancer-tissue originated spheroid method. An immortalized cell line was established by co-culture with fibroblasts. The cultured tumor cells were molecularly and functionally characterized by quantitative real-time polymerase chain reaction, Western blot, growth assay, and transwell migration assay. Results: Primary tumor cells were successfully prepared as multicellular spheroids from MNU-induced bladder tumors. The differentiation marker expression patterns observed in the original tumors were largely retained in the spheroids. We succeeded in establishing a cell line from the spheroids and named it T-MNU-1. Although basal markers (CK14 and CK5) were enriched in T-MNU-1 compared to the spheroids, T-MNU-1 expressed both luminal and basal markers. T-MNU-1 was able to migrate through a transwell. Conclusions: Tumor cells in MNU-induced bladder tumors were successfully cultured ex vivo as organoids, and an immortalized cell line was also established from them. The ex vivo models offer a platform that enables analysis of intrinsic characteristics of tumor cells excluding influence of microenvironment in MNU-induced bladder tumors.