title: Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints.
abstract: There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers' expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity.
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Terrasso
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. 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, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Margarida
authors.initials: M
authors.lastname: Serra
authors.affiliation: Medical Department, Grupo Tecnimede, 2710-089 Sintra, Portugal.
authors.forename: Augusto
authors.initials: A
authors.lastname: Filipe
authors.affiliation: Medical Department, Grupo Tecnimede, 2710-089 Sintra, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Almeida
authors.affiliation: Medical Department, Grupo Tecnimede, 2710-089 Sintra, Portugal.
authors.forename: Ana Lúcia
authors.initials: AL
authors.lastname: Ferreira
authors.affiliation: Medical Department, Grupo Tecnimede, 2710-089 Sintra, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Pedroso
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Electronic address: anabrito@itqb.unl.pt.
authors.forename: Catarina
authors.initials: C
authors.lastname: Brito
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Paula Marques
authors.initials: PM
authors.lastname: Alves
mesh_terms: D020106:Acrylamide / Q000633:toxicity; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002701:Chloramphenicol / Q000633:toxicity; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D010545:Peroxides / Q000633:toxicity; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D010545:Peroxides; D020106:Acrylamide; D002701:Chloramphenicol; C000602361:tert-butyl peroxide
keywords: 3D culture; Astrocyte; Human neurotoxicity; Neural differentiation; Neurospheres