3D Spheroid Culture Workflow using iPSC-induced Human Neurons

Human induced pluripotent stem cells (iPSC) derived neurons are now considered a more relevant in vitro model system for psychiatric and neurological diseases. They can be used for the development of physiological cell models, human disease models, and drug library screening. Three dimensional (3D) cultures are also being pursued as a more physiologically relevant system because they provide a microenvironment, cell-to-cell interactions, and biological processes that better represent in vivo conditions. The implementation of 3D spheroid culture plays an important role as an alternative approach for drug development and therapeutic applications in central neural system (CNS) disorders. To develop a neuronal 3D spheroid culture system, we tested iPSC-derived human motor neurons and cortical glutamatergic neurons using the S-BIO PrimeSurface Ultra Low Attachment Micoplates. After 2 hours, plated neurons started to settle down at the bottom of the well and form large clusters. On day 3, 3D spheroids could be seen clearly under phase contrast. On day 7, the spheroids were more condensed. The size of the 3D spheroids was proportional to the number of neurons seeded per well. In addition to the morphological assessments, a cytotoxicity assay and MEA assay were performed to demonstrate the suitable of 3D spheres as a platform for various applications. In both assays, the spheroids yielded expected results. The results presented here demonstrate the feasibility of generating uniform and reproducible spheroids using human neurons and the potential application for neurotoxicity studies.