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BACKGROUND Induced pluripotent stem cells (iPSCs) can be differentiated into virtually every desired cell type, offering significant potential for modeling human diseases in vitro. A disadvantage is that iPSC-derived cells represent an immature, which presents a major limitation for modeling age-related diseases such as Alzheimer's disease. Evidence suggests that culturing iPSC neurons in a 3D environment may increase neuronal maturity. However, current 3D cell culture systems are cumbersome and time-consuming. NEW METHOD We cultured iPSC-derived excitatory neurons in 3D precast hydrogel plates and compared their maturation to 2D monolayer cultures. COMPARISON WITH EXISTING METHODS In contrast to other hydrogel-based 3D culture techniques, which require full encapsulation of cells, our hydrogel allows the seeded iPSCs and iPSC neurons to simply infiltrate the gel. RESULTS IPSC-neurons grew to a depth of 500µm into the hydrogel. Cell viability was comparable to 2D cultures over the course of three weeks, with even better neuronal survival in 3D cultures at the one-week time point. Levels of neuronal and synaptic maturation markers, namely, neural cell adhesion molecule 1 (NCAM1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2, were strongly increased in 3D cultures. Furthermore, we identified 4-repeat (4R) tau in 3D cultures, which was not detectable in 2D cultures. CONCLUSIONS We describe a simple, hydrogel-based method for 3D iPSC culture that can serve as a fast and drug-screening-compatible platform to identify new mechanisms and therapeutic targets for brain diseases. We further provided evidence for the increased maturation of iPSC neurons in a 3D microenvironment.