As shown by AstraZeneca in nature reviews*, one third of safety failures along the drug discovery process is linked to CNS toxicity uncovered in clinical trials. To avoid this attrition, the potential neurotoxicity of any drug going through the blood brain barrier (BBB) needs to be assessed in the very early stages of new chemical entities (NCE) research. Neurotoxicity assays can be performed on the SH-SY5Y human cell line by using High-Content Screening (HCS) technologies. The present study was performed using classical 2D and 3D culture protocols. In this poster, 2D results and preliminary 3D culture results on multiple reference compounds are depicted.

1. Neurotoxicity assay on 2D and 3D culture using
High Content Screening (HCS) technology
K. Jarnouen1, G. Frugier2*, C Point, J. Bursztyka1 and N. Maubon1*
1 HCS Pharma, Bât A, 250 rue Salvadore Allende, 59120 Loos
2Molecular Devices, 660 – 665 Eskdale Road, Winnersh Triangle, Wokingham, Berkshire, RG41 5TS, United Kingdom
1*nathalie.maubon@hcs-pharma.com 2* guillaume.frugier@moldev.com
Abstract
As shown by AstraZeneca in nature reviews*, one third of safety failures
along the drug discovery process is linked to CNS toxicity uncovered in
clinical trials. To avoid this attrition, the potential neurotoxicity of any drug
going through the blood brain barrier (BBB) needs to be assessed in the
very early stages of new chemical entities (NCE) research. Neurotoxicity
assays can be performed on the SH-SY5Y human cell line by using High-
Content Screening (HCS) technologies. The present study was performed
using classical 2D and 3D culture protocols. In this poster, 2D results and
preliminary 3D culture results on multiple reference compounds are
depicted.
Methods
Results
Conclusions & Perspectives
✓ First experiments on 3D culture show similar results compared to 2D culture for all tested compounds, at the exception of
colchicine.
✓ Colchicine seemed to have higher toxicity effect on 3D culture compared to 2D culture. For the experiments in 3D, cells
count can be performed in the same well with the 2D (around the clump) and on 3D (in the clump). Analysis demonstrate
the different effect between 2D and 3D.
➢ Perspectives : In order to verify the impact of 3D culture compared to 2D culture, a bank of different neurotoxic and non
neurotoxic compounds will be perfomed on this assay. Furthermore, 3D analysis can also be improved by analyzing the
length of neurites in and around the clump.
Cell culture: neuronal cells (SH-SY5Y) were routinely maintained in MEM/F12 (v/v) supplemented
with 10% serum. Neuronal cells were seeded at 10 000 or 100 000 cells/well in 96-well plates
(Greiner® advanced TC µClear or Greiner® medium binding) in MEM/F12 supplemented with
differentiation agent for 2D or 3D culture, respectively. Then, cells were incubated at 37 °C in 5 % CO2
for 3 days for plating and differentiation.
Treatment assay: Neurotoxicity assay was performed. Medium was changed in each well by fresh
medium added with 8 compounds tested at different concentrations. After 48h of incubation, cells
were incubated with mitotracker during 30 minutes, fixed and then stained with b III tubulin and
Hoechst. Image acquisition was performed on ImageXpress Micro confocal (Molecular Devices) and
the analysis of cell number and neurite length in 2D or 3D spheroid volume was done through
MetaXpress software (Molecular Devices) by using 3D analysis module. This assay can be performed
on our complete automated platform set up in Lille (France).
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* Cook D, Brown D, Alexander R, March R, Morgan P, Satterthwaite G, Pangalos MN (2014) Lessons learned from the fate of AstraZeneca’s drug pipeline: a five-imensional framework. Nat Rev Drug Discov. 13(6):419-31.
Hoechst
bIII-tubulin
Mitotracker
2D culture 3D culture
0
20
40
60
80
100
120
140
0.003 0.01 0.03 0.1 0.3 1 3 10 30 100
[Methylmercure] (µM)
Cells in 3D
volume
(%/CTRL)
Neurite
intensity in 3D
volume
(%/CTRL)
0
20
40
60
80
100
120
0.003 0.01 0.03 0.1 0.3 1 3 10 30 100
[Colchicine] (µM)
Cells in 3D volume (%/CTRL)
Neurite intensity in 3D volume (%/CTRL)
CTRL cells Me-mercure
0
20
40
60
80
100
120
140
0.003 0.01 0.03 0.1 0.3 1 3 10 30 100
[Methylmercure] (µM)
Cell count
(%/ctrl)
Neurite
length per
cell (%/ctrl)
0
20
40
60
80
100
120
0.003 0.01 0.03 0.1 0.3 1 3 10 30 100
[Colchicine] (µM)
Cell count (%/ctrl)
Neurite length per cell (%/ctrl)
Lead
2D
3D 0
20
40
60
80
100
120
140
0.003 0.01 0.03 0.1 0.3 1 3 10 30 100
[Colchicine] (µM)
2D nuclei count (%/CTRL)
3D nuclei count (%/CTRL)
2D & 3D cell counts on the
3D experiment
Different Z stacks
in the clump
(Δz = 6 µm)
3D reconstruction
Maximum
projection from 30
stacks