Fluorescent biosensor-based functional validation of biofabricated 3D human iPSC-derived neuronal models for drug screening

Authors

• KF Fuchs
• JA Sommers
• M Neog
• F Miller
• L Rider
• R Goldbach-Mansky
• A de Jesus Rasheed
• RM Brosh Jr

Abstract

More than 90% of the drugs entered clinical trials fail because of lack of efficacy or unexpected toxicity. This high failure rate partly attributed to the use of in vitro cellular assays and animal models that do not reproduce human physiology and pathology during preclinical drug development. We develop 3D organotypic cellular models that capture native-like physiological features of tissues and serve as assay platforms for diseases and predictive therapeutics development and to study neurological and neurodegenerative diseases. We used fibrinogen gel with gelatin and laminin, a 3D extracellular matrix that enables the co-culture of human iPSC derived neurons and astrocytes to autonomously form neuronal networks and spontaneous synaptic connections with a native-like neuronal density thus mimicking the dynamics of human functional brain circuitry. For the functional assays, we performed double AAV-transfections of ChrimsonR-opsin with combination of different biosensors, GCaMP6f, dLight1.2 and iGluSnFr, for real time calcium flux measurement, released neurotransmitter like dopamine, glutamate assessment, respectively. The measurements of different biosensors from the functional 3D model, were modulated by optogenetics stimulation, and perturbed by pharmacological agents in a manner to be expected from in vivo data. This model can be produced in a 384/96-well platform for therapeutic screening. The fibrinogen-gel used here is compatible with extrusion-based bioprinting, should enable the biofabrication of spatially defined functional neuronal circuits. Furthermore, the approach allows for gradual inclusion of additional physiological complexity, including vasculature, additional cells like microglia in flexible 3D bioprinted patterns to mimicking various human brain region selective circuitry.

Scientific Focus Area: Neuroscience

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