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Quantitative live-cell analysis for optimization of culture conditions and evaluation of cell health in human induced pluripotent stem cell-derived neurons

A major limitation in studying human diseases affecting the nervous system is the ability to culture, monitor and analyze neuronal cells that accurately represent human phenotypes of these disorders. The use of human induced pluripotent stem cell (hiPSC)-derived neurons has provided a new approach aimed at modeling neurological diseases. Monitoring neuronal morphology and cell health in long-term culture is critical for the characterization and evaluation of these novel model systems. Traditional approaches rely on endpoint assays and imaging techniques that require immunochemical staining, which lacks real-time kinetic information.

This presents a liability when studying models of the nervous system where the integrity of neural networks is compromised and the dynamic complexity of neurological responses and features are lost when utilizing these types of approaches. For these model systems, continual real-time monitoring offers a significant advantage in that it provides a more physiologically relevant picture of neuronal cell behavior, allows for non-invasive, repeated measurements of the same neuronal networks over time, and enables the capturing of rare or transient events that are often missed with end-point assays.

In this application note, we describe methods and present validation data highlighting optimal culture conditions for evaluation of cell viability and neurite outgrowth in hiPSCderived neurons from Cellular Dynamic International (CDI, iCell Neurons). We also monitor neurite outgrowth and cellular viability in iCell Gluta Neurons from CDI using a quantitative, live-cell imaging and analysis approach with the IncuCyte® S3 over days/weeks in 96-well microplate culture. To exemplify a real-time imaging and analysis approach using hiPSCderived neurons, we assess neuronal excitotoxicity using the IncuCyte® S3 Phase/Fluorescent NeuroTrack applications multiplexed with Annexin V reagents. These assays outline optimal culture conditions for an example iPSC-derived neuronal system and demonstrate the ability of the IncuCyte approach for real-time, long-term quantitative analysis of iPSC-derived neuronal cell health.