title: Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models.
abstract: Currently there is an effort toward the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000-1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Vítor E
authors.initials: VE
authors.lastname: Santo
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Marta F
authors.initials: MF
authors.lastname: Estrada
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Sofia P
authors.initials: SP
authors.lastname: Rebelo
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Sofia
authors.initials: S
authors.lastname: Abreu
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Inês
authors.initials: I
authors.lastname: Silva
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Pinto
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Susana C
authors.initials: SC
authors.lastname: Veloso
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Ana Teresa
authors.initials: AT
authors.lastname: Serra
authors.affiliation: AbbVie Inc., North Chicago, IL 60064, USA.
authors.forename: Erwin
authors.initials: E
authors.lastname: Boghaert
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal. Electronic address: anabrito@itqb.unl.pt.
authors.forename: Catarina
authors.initials: C
authors.lastname: Brito
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor / Q000166:cytology*; D049109:Cell Proliferation; D002470:Cell Survival; D018920:Coculture Techniques; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D057446:Hydrodynamics; D008954:Models, Biological; D015203:Reproducibility of Results; D018874:Spheroids, Cellular / Q000166:cytology*
keywords: 3D cell culture; Drug discovery; In vitro cancer models; Stirred-tank culture systems; Tumor spheroids