• Neurotoxicity is one of the major causes for adverse drug reactions and drug attrition during preclinical or clinical development, accounting for approximately 10% of all drug withdrawals regardless of therapeutic area [1].
  • This highlights the importance of improving risk assessment early in the drug discovery process and demonstrates the limitations of current animal models, which fail to clearly identify the risks of drug induced neurotoxicity in humans.
  • “Successful drug development processes should involve the implementation of in vitro approaches that can be utilized to address specific mechanistic questions around theoretical or identified neuropharmacological effects, whether detected during the development process or after approval. In vitro assays can also be used as screening tools to improve the chemical content and mitigate the potential neurotoxicity of future drug candidates, help to limit the number of compounds that are tested in vivo, reduce time and costs associated with neurotoxicity testing, and ultimately limit the impact that safety issues have clinically and with regard to withdrawal due to safety issues related to neurotoxicity.” [1]
  • Mechanistic neurotoxicity can be evaluated in vitro through the use of cultured neuronal models in traditional monolayer assay format, or with 3D neuronal organoids.
  • Assessment of neurotoxicity can be accomplished by assessment of cytotoxicity to neurons, activation of astrocytes, and evaluation of modulation of calcium signaling.


InstrumentMolecular Devices ImageExpress Micro Confocal
Analysis MethodHigh content screening
MarkersLIVE/DEAD fixable dead cell probe (Molecular Probes)
DAPI (total cell count)
Ca+2 probe
Other markers available on request
Cell Model Type3D cell models (e.g. tumor spheroids, organoids, etc.)
or adherent monolayer / cell suspension
Cell Types AvailableNeuronal monolayer format
iPSC derived neural organoid (Stemonix)
Custom models available on request
Test Article Concentration8 point assay (0.05, 0.1, 0.5, 1, 5, 10, 50, 100 µM)
(custom concentrations available)
Single point assay
Number of Replicates3 replicates per concentration
Quality Controls0.5% DMSO (vehicle control)
Domoic acid (positive control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryDose response curves, AC50 values, total cell counts, Viability % for each test concentration
Evaluation of statistical significance of results with respect to vehicle and positive control

General Procedure

  1. For 3D cell models, grown to approximately 500 μm in diameter. Adherent monolayers cultured to confluence.
  2. Treatment with test compounds
  3. After 24 hours, cells are labeled with fixable dead cell probe, Ca+2 probe, and immunolabeling for GFAP or other markers
  4. For 3D cell models, tissue clearing is applied to render 3D cell models transparent
  5. High content imaging is conducted on well plates
  6. Images are analyzed to quantify total number of cells, total number of dead cells, total number of astrocytes, astrocyte activation, alterations to calcium signaling

Representative Data

Figure 1. Maximum Z-projection of Z-stack obtained from High Content Imaging of iPSC derived neuronal organoid depicting astrocytes stained with GFAP (red), dead cells (green), and nuclei (blue).

Figure 2. Representative data showing the observed fraction of dead cells detected in primary tumor spheroids treated with various cytotoxic agents

Neuronal Organoid Comparison

Figure 3. Z-Projection Image of microBrain® 3D cortical spheroid labeled with anti-Glutamine Synthetase (green) counterstained with DAPI (blue).

Figure 4. Quantification of glutamine synthetase expression in treated and untreated neuronal organoids


  1. Staflin, K., Misner, D., & Dambach, D. (2016). Utilization of In Vitro Neurotoxicity Models in Pre‐Clinical Toxicity Assessment. Stem Cells in Toxicology and Medicine, 155-178.