High‐Throughput Neurite Outgrowth Assay Using GFP‐Labeled iPSC‐Derived Neurons

The potential neurotoxicity from an increasing number of drugs and untested environmental chemicals creates a need to develop reliable and efficient in vitro methods for identifying chemicals that may adversely affect the nervous system. An important process in neurodevelopment is neurite outgrowth, which can be affected by developmental neurotoxicity. Currently, neurite outgrowth assays rely mainly on staining, which requires multiple sample processing steps, particularly washing steps, that may introduce variation and limit throughput. Here, we describe a neurite outgrowth assay that uses induced pluripotent stem cell (iPSC)–derived human cortical glutamatergic neurons and/or spinal motor neurons labeled with green fluorescent protein (GFP) to test compounds in a high‐content and high‐throughput format. This method enables live and time‐lapse imaging of GFP‐labeled neurons using an assay plate that is continuously imaged at multiple times after chemical treatment. In this article, we describe how to thaw frozen GFP‐labeled neurons, culture them, treat them with a compound of interest, and analyze neurite outgrowth using a high‐content imaging platform. In this assay, GFP‐labeled iPSC‐derived human neurons represent a promising tool for identifying and prioritizing compounds with potential developmental neurotoxicity for further hazard characterization. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. This article was corrected on 23 September 2022. See the end of the full text for details. Basic Protocol 1: Thawing and seeding of iPSC‐derived neurons Basic Protocol 2: Compound plate preparation and treatment of neurons Basic Protocol 3: High‐content imaging and analysis