Development of co-culture Parkinson’s diseasemodel using High Content Screening technology

N. Maubon1, J. Bursztyka1, and V. Racine2

1nathalie.maubon@hcs-pharma.com ; 2victor.racine@quantacell.com

AbstractParkinson's disease (PD), adult neurodegenerative disorder, is caused by the death of dopamine neurons in the substantia nigra. High contentscreening (HCS) should allow finding new pathways involved in the onset of PD by screening molecules based on death phenotype. Rotenone,a chemical compound commonly used as pesticide, is well-documented as death inducer of dopamine neurons in the substantia nigra andallow to mimic PD in vitro and in vivo. Rotenone-induced degeneration of dopaminergic neurons may not be solely attributed to animpairment of neuronal mitochondrial complex I activity in the dopaminergic neurons but may also be boosted by the participation of theresident immune cells in the brain: the microglia cells. Effectively, various environmental factors may also work in concert to inducedopaminergic neurodegeneration and numerous studies have confirmed that neuroinflammation plays a critical role in the pathogenesis ofneurodegenerative deseases, including PD.To develop this assay, human neuroblastoma and mouse macrophagic cell lines in coculture were used, and rotenone was chosen as deathinducer and phenotypic markers as Hoechst for nucleus and MAP2 for neurites analysis.Furthermore, first assays in 3D cell co-culture show promising results and give us nice perspectives for the future.

MethodsResults

Conclusions & Perspectives

A nice dose toxicity effect of rotenone on coculture cells (neuronal and macrophages cells) was observed on cell deathand on neurite length with EC50 at around 0.2 µM for both.

Neuroprotective effect on cell death induced by rotenone 1 µM in a context of neuroinflammation in high contentscreening allows identifying new compounds and new pathways for PD treatment (assay was performed with K252a, ASK1inhibitor: data not shown).

First results on 3D cell culture on biomimesys® plates from CELENYS show that neuronal and macrophagic cells have twodifferent behaviors in culture: SH-SY5Y cells form spheroids while macrophagic cells stay separated.

Perspective: This model will be developed in 3D culture when differentiation of neuronal cells in 3D culture will beverified.

Cell culture: neuronal cells (SH-SY5Y) and macrophage cells (Raw264.7)were routinely maintained in MEM/F12 (v/v) supplemented with 10%serum. Neuronal cells were seeded alone or in co-culture withmacrophage cells at 10 000 cells/well in 96-well Corning cellBind plates inMEM/F12 supplemented with differentiation agents. Then, cells wereincubated at 37 °C in 5 % CO2 for 3 days for plating and differentiation.

Treatment assay on 2D culture: Parkinson induction assay was performedby replaced medium in each well by fresh medium with rotenone atdifferent concentrations with or without LPS. After 48h of incubation, cellswere fixed and stained with MAP2, and Hoechst. Image acquisition wasperformed on ImPACcell platform (Arrayscan) and analysis was donethrough Columbus (Perkin).

3D Cell culture: neuronal cells and macrophage cells were seeded alone orin coculture at 25 000 cells/well in Biomimesys® plate from CELENYS inMEM/F12 supplemented with differentiation agents. Then cells wereincubated at 37 °C in 5 % CO2 for 3 days for plating and differentiation.Analysis and visualization were obtained using innovative algorithms andsoftware solutions dedicated to 3D cell culture. It allows the classificationof cell phenotypes and the measurement of 3D cell organization.

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CTRL cells Rotenone (30 µM) treated cells

Rotenone treatment on 2D coculture

Rotenone treatment on 2D coculture3D co-culture of neuronal and macrophage cells

Neuronal cells Macrophage cellsNeuronal &

Macrophage cells

After 48H of incubation in Biomimesys® plates, magnitude: 40X

After 10 days of incubation in Biomimesys® plates, stained with Hoechst (blue) & MAP2 (green)

3D co-culture of neuronal and macrophage cells

Neuronal cells Macrophage cellsNeuronal &

Macrophage cells

rotenone CTRL

100 µM 30 µM 10 µM 3 µM 1 µM 0,3 µM 0,1 µM 0,03 µM 0,01 µM 0,003 µMDMSO0,5%

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