The use of iPSCs for modeling neurological disorders addresses a critical need in medicine, yet technical limitations in neuronal cultures have made the analysis of individual neurons and neuronal networks very challenging. The clustering of human neurons during the differentiation process makes it difficult to image single cell bodies and to track the fates of individual neurons over time. The challenge, in particular, of culturing human neurons in a micropatterned environment is in providing conditions that ensure survival of single cells on a cell-adhesive site as well as to maintain the defined pattern during long-term neuronal culture. In this work, we demonstrate that the use of patterned monolayers overcomes limitations such as neuronal clustering and enables the long-term culture of human neurons under well-defined conditions. We give examples of the use of these patterned cultures in the quantitative tracking of mitochondrial movement, evaluation of mitochondrial morphology, analysis of neurite outgrowth and neuronal polarity in specific subtypes of neurons. These applications have not been possible with the use of conventional iPSC culture formats, and serve to illustrate the significance of the work presented in this paper.
