title: Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.
abstract: BACKGROUND
  Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches.
  
  
  METHODS
  In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated.
  
  
  RESULTS
  Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified.
  
  
  CONCLUSIONS
  The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates.
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Terrasso
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
authors.forename: Ana Carina
authors.initials: AC
authors.lastname: Silva
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: Pedro
authors.initials: P
authors.lastname: Pedroso
authors.affiliation: Medical Department, Grupo Tecnimede, 2710-089 Sintra, Portugal.
authors.forename: Ana Lúcia
authors.initials: AL
authors.lastname: Ferreira
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
authors.forename: Paula Marques
authors.initials: PM
authors.lastname: Alves
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal. Electronic address: anabrito@itqb.unl.pt.
authors.forename: Catarina
authors.initials: C
authors.lastname: Brito
mesh_terms: D001253:Astrocytes / Q000166:cytology / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D018696:Neuroprotective Agents / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects
chemical_list: D018696:Neuroprotective Agents
keywords: 3D co-culture; Astrocyte; Human neurotoxicity; In vitro assay; Neuron; Neuroprotection