Spheroid-based cellular platforms are considered to enable more complex, biologically relevant, and predictive assays for compound screening, safety evaluation and toxicity studies. Thus, here we deployed a high throughput spheroid co-culture of cortical glutamatergic and GABA-ergic neurons as well as astrocytes, more closely resembling the tissue constitution of native human brain tissue. Whole-genome RNAseq profiling demonstrated neural tissue expression patterns with high content imaging validating neuronal and astrocytic cell populations while showing highly reproducible spheroid size across 96 and 384-well plates. Functional neuronal circuitry was confirmed with MEA recordings and visualized under high-throughput conditions as robust spontaneous, synchronized calcium oscillations with reproducible baseline activity patterns across wells and plates.
In order to validate the capabilities of the platform for compound profiling and discovery, a library of 1622 FDA approved compounds was screened in a single point at 10μM final concentration using Ca2+ oscillations as a functional phenotypic readout. The library included drugs covering a wide spectrum of targets such as oncology, cardiology, anti-inflammatory, immunology, neuropsychiatry, and analgesia. DMSO was used as control and showed a standard deviation of 9% across all plates and a Z’ of 0.73 was observed for the whole screen. Hits were identified as responses that were at least 3 standard deviations from DMSO control responses. As expected, the highest number of hits arrived from targets associated with neuronal signaling (serotonin, dopamine, GABA and adrenergic receptors). Interestingly, several compounds leading to higher cAMP accumulation lead to an increase in peak count similar to what is noted using 4-AP, a known pro convulsant. Hit confirmation in 7 point dose-response and exploration of pathways is currently ongoing. In conclusion, high throughput functional assays using the human iPSC-derived 3D neuronal spheroids platform deployed in this study demonstrated the ability to identify a wide range of hits spanning multiple target areas. Thus this model may serve as a phenotypic and target-based platform for identifying new starting points for novel CNS discovery.