Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Neurotoxicity assay on 2D and 3D culture using High Content Screening (HCS) technology

242 views

Published on

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.

Published in: Health & Medicine
  • Be the first to comment

  • Be the first to like this

Neurotoxicity assay on 2D and 3D culture using High Content Screening (HCS) technology

  1. 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). To get this poster, please flash the QR- code You can use the I-NIGMA application from your store * 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

×