Tuneable hydrogel-based micropillar arrays for myelination studies.

Oligodendrocytes (OLs) play a critical role in central nervous system (CNS) function by myelinating axons and enabling rapid signal conduction. To model the key biomechanical parameters that regulate myelination, we developed a tuneable hydrogel-based micropillar array system that mimics the three-dimensional (3D) architecture and mechanical softness of axons. This platform supports a long-term culture of OLs and permits the formation of multilayered compact myelin by both rodent and human iPSC-derived OLs. Using fluorescence confocal imaging and transmission electron microscopy (TEM), we observed a strong linear correlation between immunostained myelin thickness and the number of myelin wraps, enabling fast, high-content quantification of myelination. By systematically varying pillar stiffness, diameter, and surface coatings, we show that both the mechanical and geometric properties of axon-like substrates critically regulate OL 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 cost-effective and scalable platform offers a physiologically relevant, high-throughput assay to dissect OL biology and accelerate the discovery of remyelinating therapies for demyelinating diseases such as multiple sclerosis. Overall design: RNA-seq profiling of oligodendrocytes isolated from P7 Sprague Dawley rats, cultured and differentiated on flat or micropillar hydrogels.