Assay Development and Screening
Advanced In Vitro Cell Culture Systems
Central nervous system (CNS)-based drug discovery has been hampered by a lack of relevant, high-throughput experimental platforms. Complex, three-dimensional (3D), experimental preparations with multiple cell types better represent the native, in vivo biology, thus providing relevant material for CNS investigations. Unfortunately, these preparations traditionally have not been able to support the throughput necessary for early-stage discovery programs. The ideal preparation would provide consistent native tissue function in high throughput plates. To meet this need, we have developed 96- and 384-well assay-ready, 3D neural spheroid platforms; each spheroid is composed of cortical glutamatergic and GABA-ergic neurons co-cultured with astrocytes to provide a more complex, biologically relevant, and predictive preparation in a high throughput platform for compound screening, safety evaluation, and toxicity studies.
Whole genome RNAseq profiling demonstrated neural tissue expression patterns, and high content imaging validated neuronal and astrocytic cell populations while showing highly reproducible spheroid size across both 96 and 384-well platforms. Functional neuronal activity was confirmed with MEA recordings and visualized under high-throughput conditions as robust spontaneous, synchronized calcium oscillations with consistent and reproducible baseline activity patterns across wells and plates. Functional circuitry was confirmed by challenging the system with specific ion channel and neurotransmitter receptor agonists and antagonists.
To validate the capabilities of the platform for compound profiling and discovery, a library of 1622 FDA approved compounds was screened in single point at 10 μM final concentration examining Ca2+ oscillations as a functional phenotypic readout. The library included drugs covering a wide spectrum of targets such as CNS biology, oncology, cardiology, anti-inflammatory, immunology, neuropsychiatry and analgesia with DMSO as a vehicle control. Hits were identified as responses that were at least 3 standard deviations from DMSO control responses. As expected, the highest number of hits were from targets associated with neuronal signaling (serotonin, dopamine, GABA, and adrenergic receptors), neural biology, and second messengers such as cAMP. Of note was the identification of several compounds that led to increases in peak count similar to that of 4-AP, a known pro-convulsant. The results validated a robust screening platform with a vehicle control standard deviation of ~9% across all plates and a Z’ score of 0.73 across the entire screen.