Tunable hydrogel-based micropillar arrays for myelination studies.

Oligodendrocytes enable rapid central nervous system signaling by myelinating axons. Here, to model key biomechanical cues regulating myelination, we developed a tunable hydrogel-based micropillar array system that mimics the three-dimensional architecture and softness of axons. This platform supports the long-term culture of oligodendrocytes and robust formation of multilayered compact myelin by rodent and human oligodendrocytes. Using confocal and transmission electron microscopy, we observed a strong linear correlation between immunostained myelin thickness and the number of myelin wraps, enabling high-content quantification of myelination. Systematic variation of pillar stiffness, diameter and surface chemistry within pathophysiological ranges revealed that both mechanical and geometric properties of axon-like substrates critically regulate oligodendrocyte differentiation and myelin wrapping. Importantly, we demonstrate that pharmacological agents exhibit stiffness-dependent effects on myelination, suggesting that overly rigid in vitro models may yield false-positive drug hits. This platform offers a physiologically relevant, high-throughput assay for dissecting oligodendrocyte biology and discovering remyelinating therapies for diseases such as multiple sclerosis.