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3D Neurotoxicity Assay

Detect neurotoxicity of novel therapeutics with enhanced in vivo relevance using Cyprotex’s 3D brain microtissue combined with high content imaging (HCI) end points.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Background Information

  • The prevalence of adverse neurotoxic reactions of the brain in response to drugs or environmental hazards continues to prompt the development of novel cell-based assays for accurate neurotoxicity prediction1.
  • In vitro three-dimensional cell cultures allow better recapitulation of the complex in vivo microenvironment than traditional 2D monolayer models2.
  • 3D models also permit long-term compound exposures allowing a closer replication of clinical dosing strategies3
  • Mitochondrial dysfunction and calcium homeostasis4 are commonly observed responses to toxic compounds and are implicated in neurotoxicity
  • Confocal high content imaging (HCl) allows the simultaneous detection of multiple cell health parameters within a 3D microtissue structure in combination with a measure of cellular ATP content
HCA can be used to precisely distinguish between neuron-specific toxicity and general cytotoxicity while simultaneously enabling inclusion of other parameters to detect novel neurotoxic effects of chemicals.

1Wilson MS et al., (2014) NeuroToxicology 42; 33-48


3D Neurotoxicity Protocol

Microtissue Induced pluripotent stem cell (iPSC) derived neurocytes and astrocytes
Analysis Platform Confocal Cellomics ArrayScan® XTI or
CX7 (Thermo Scientific)
Test Compound Concentrations 8 point dose response curve with top concentration based on 100x Cmax or solubility limit
3 replicates per concentration*
Compound Requirements Maximum (dependent upon number of repeat doses) 150 μL of a DMSO* solution to achieve 200x top concentration maintained at 0.5% DMSO or equivalent amount in solid compound
Time Points Any time point up to 14 days
Quality Controls Negative control: 0.5% DMSO (vehicle)
Positive control: chloroquine and colchicine
Data Delivery Minimum effective concentration (MEC) and AC50 value for each measured parameter; microtissue count, microtissue size, DNA structure (DNA), calcium homeostasis (Ca2+) mitochondrial mass (Mito Mass), mitochondrial membrane potential (MMP) and cellular ATP content (ATP)*

* other options available on request.


Data from Cyprotex's 3D Neurotoxicity Assay


Figure 1
Representative 3D confocal high content screening (HCS) images of brain microtissues labelled with Hoechst (Blue; DNA structure and microtissue size), Fluo-4 AM (Green; calcium homeostasis) and TMRE (Red; mitochondrial function) following exposure to either control DMSO or effective concentrations of known neurotoxins chloroquine, colchicine and lead acetate. 
Table 1
Neurotoxicity prediction of 10 reference compounds categorised according to literature data and normalized to total plasma Cmax in immature and matured brain microtissues.

Immature (3 day old) or matured (14 day old) brain microtissues were exposed to test compound for 72 or 336 hrs. During the 336 hr period re-dosing occurred on 3 occasions (72, 168 & 210 hrs. At either 72 or 336 hr the cell model was analysed using the confocal mode of Cellomics ArrayScan® XTI or CX7 (Thermo Scientific) following incorporation of fluorescent dyes. Cellular ATP content was subsequently measured using CellTiterGlo® (Promega).

Figure 2
Graphical representation of early calcium dyshomeostasis followed by a decrease in microtissue size in response to colchicine following (a) 72 hours and (b) 336 hours in brain microtissues.

Utilising the 3D neurotoxicity assay approach 80% of reference compound toxicities were correctly predicted within a 100x Cmax cut off with a 336 hour exposure period in both immature and matured brain microtissues. Following the acute time point of 72 hour compound exposure, only 50% and 60% of compounds were correctly predicted within a 100x Cmax cut off in the immature and matured brain microtissues, respectively.

An in vitro 3D brain microtissue model with improved longevity and better recapitulation of in vivo cellular physiology in combination with an automated multiparametric HCI and a cytotoxicity assay presents a viable screening strategy for the accurate in vivo relevant detection of novel therapeutics with neurotoxicity potential early in drug development.


1 Wilson MS et al., (2014) Multiparametric high content analysis for assessment of neurotoxicity in differentiated neuronal cell lines and human embryonic stem cell-derived neurons. Neurotoxicology 42; 33-48
2 Anderl JL et al., (2009) A neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity. J Vis Exp 27; e1173
3 Pamies D et al., (2017) A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity. ALTEX 34(3); 362-376.
4 Guo GW & Liang YX (2001) Aluminium-induced apoptosis in cultured astrocytes and its effect on calcium homeostasis. Brain Res 888; 221-226

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