Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds

Ana Paula Terrasso, Ana Carina Silva, Augusto Filipe, Pedro Pedroso, Ana Lúcia Ferreira, Paula Marques Alves, Catarina Brito

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)


Introduction 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. Discussion 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.

Original languageEnglish
Pages (from-to)72-79
Number of pages8
JournalJournal of Pharmacological and Toxicological Methods
Publication statusPublished - 1 Jan 2017


  • 3D co-culture
  • Astrocyte
  • Human neurotoxicity
  • In vitro assay
  • Neuron
  • Neuroprotection


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