Development of CNS model for neurodegenerative drug discovery using 3D printed human iPSC-derived cells Matthieu Trigano, Anna-Lena Zepernick, Eve Corrie, Emma V. Jones Medicines Discovery Catapult, Block 35, Alderley Park, Cheshire, SK10 4ZF, UK
Introduction
There is a lack of effective medicines for the treatment of neurodegenerative diseases. This challenge is also coupled with a lack of validated preclinical models, which is limiting drug development and successful progression to clinical trial. Over the past decade, considerable effort has been devoted to improving in vitro preclinical models of the central nervous system (CNS) to more accurately replicate brain structure and function. One promising direction includes the development of complex 3D models such as bio-printed models which more closely mimic the intricate cell-cell and cell-extracellular matrix interactions observed in vivo. Increasing evidence suggests that 3D models more effectively reproduce the pathological processes of neurodegenerative diseases when compared to traditional 2D systems, making them potentially more predictive and valuable for translational drug discovery.
Methods
Tri-culture Neurons/Astrocytes/Microglia
Microglia
Co-culture Neurons/Astrocytes
Optimizing and Characterizing 3D Co- and Tri- Cultures Calcein labelling of neuron-astrocyte cocultures at multiple time points showed robust cell viability and increased culture complexity over time.
200µM
1 week
4 week
5 week
6 week
7 weeks
8 week
To distinguish neurons from astrocytes, co-cultures were transduced with NeuroLight lentivirus, encoding an mRuby fluorescent protein driven by the synapsin promoter. These results demonstrate that the hydrogel matrix is amenable to efficient transduction by lentiviruses.
20µM 100 μM
100 μM
Calcein
100 μM
NeuroLight
Calcein / NeuroLight
Live TMRM imaging provides a qualitative readout of mitochondrial membrane potential (ΔΨm) in 3D neuron–astrocyte cultures.
Induction of neuroinflammation in 3D CNS Tri-cultures GFP Microglia GFAP βIII-tubulin
Conclusion • We have successfully demonstrated that a 3D model of neurons and astrocytes are able to grow and remain viable for up to 8 weeks. • The 3D model is amenable for both fixed and live cell imaging (e.g. viability, neuronal activity, mitochondrial function).
10µM
LPS and Aβ42 induced microglia-dependent IL-6 release. LPS + Nigericin triggered a robust IL-1β secretion which is consistent with inflammasome activation. There is no detectable IL-6 in LPS-stimulated cultures lacking microglia confirming microglia as a primary source of inflammatory cytokines. Using immunocytochemical methods and confocal imaging, we observed microglia (GFP) in close proximity of neurons (β3-tubulin) and astrocytes (GFAP).
• We have established a CNS triculture model in which microglia are invading the gel and migrating towards neurons and astrocyte. • Using different stimuli, we have shown that 3D CNS tricultures can serve as a model to investigate neuroinflammation.