Characterization of primary three-dimensional cell culture from spontaneous canine tumors.

This study aimed to generate primary three-dimensional (3D) cell cultures derived from spontaneous canine tumors and to compare two culture systems: a ready-to-use bioreactor (VITVO®) and a miniaturized plate-based system (MPBS). A total of 27 tumors, including mainly hemangiosarcomas, oral melanomas, urothelial carcinomas, and osteosarcomas, were collected from three Italian veterinary hospitals. Fresh samples were used to establish 3D cell cultures and to perform histological and immunohistochemical analyses for phenotypic characterization and comparison with primary tumors. Tissue dissociation proved to be a critical step. A manual protocol combining mechanical and enzymatic methods significantly improved cell viability and cellularity compared to automated dissociation, which yielded fewer viable single cells and limited cell survival. Both culture systems successfully supported 3D cell cultures using the same scaffold material. The plate-based system was particularly suitable for small biopsy samples without compromising cellularity, whereas VITVO® was more appropriate for larger tissue specimens. An inverse correlation was observed between the extent of necrosis in primary tumors and the cellularity of the corresponding 3D cell cultures. Immunohistochemical analyses confirmed that most 3D cell cultures retained the phenotypic characteristics of the original tumors, while osteosarcoma models exhibited limited cell maintenance, likely due to stromal predominance. Overall, these findings highlight the importance of optimizing dissociation protocols, scaffold size, and culture conditions to preserve tumor heterogeneity and viability. This workflow provides a platform for generating 3D models with preserved morphological and immunophenotypic concordance with the primary tumors.