DEVELOPMENT OF A SINGLE-CELL RESOLUTION, HIGH-CONTENT SCREENING PLATFORM FOR REAL TIME TRACKING OF NEURAL SPHEROID HEALTH

Most drug screening protocols for neurodegenerative diseases rely on simple 2D cultures, which lack the cellular complexity of the brain microenvironment. 3D organoid models offer greater physiological relevance, but present challenges for reliable, high-throughput imaging with cellular resolution. We aimed to develop a high-content imaging platform, with single-cell resolution for monitoring neural health in live 3D brain spheroids over time. Induced pluripotent stem cells were patterned to a cortical progenitor identity and matured for 100 days in 3D to model aspects of the human cortex, including layer specification and glial cell integration. We sought to validate live cell dyes for apoptosis (CellEvent Caspase 3/7) and reactive oxygen species (CellROX), two key hallmarks of neurodegeneration, under oxidative stress (50 or 200 μM sodium arsenite). Confocal imaging was automated over the course of 72 hours, with timepoints every 4 hours, utilising the Zeiss Cell Discoverer 7. We observed a dose-dependent decrease in viability and increase in reactive oxygen species with a large number of cortical spheroids at whole spheroid and single-cell levels. In a preliminary screen we also demonstrate pharmacological protection or exacerbation of these phenotypes using neuroprotective compounds and environmental toxins, respectively. In summary, we have developed a live cell imaging platform for cortical spheroids, as a robust system for high-throughput screening of compounds that impact neural health.

This platform will enable us to phenotype disease models and identify compounds that reduce pathological phenotypes, providing a more clinically relevant screening approach for neurodegeneration.