Over the past decades, many studies have explored the cellular and molecular mechanisms that underlie neuronal polarization primarily using mouse primary neurons. Also, iPSC-derived neurons from genetic models of neurodegenerative diseases have been used to study disease-associated changes of neuronal organization or network formation, primarily focusing on neurite outgrowth phenotypes [36-37]. Further, high throughput screening studies for human neurons have been developed to assess phenotypic readouts such as neurite outgrowth and branching and suggested its use for compound screening as well as drug development [38]. However, it has been very challenging to reliably examine these processes in iPSC-derived human neurons under conventional culture conditions due to aforementioned clustering of cells. There have been attempts to culture non-iPSC-derived cells in microfluidic platforms to establish a more organized environment for studies of neurite outgrowth, but those platforms are designed to guide the neurites towards a predetermined direction. In addition, this approach has not been utilized in iPSC-derived neurons [52]. We found that neurons plated on star-shaped micropatterned substrates initially formed individual connection to neighboring cell bodies either by the axon or the
