Elastomeric 3D‐Printed Microenvironments Enable Nanonewton Force Measurements in Healthy and Diseased Human Pluripotent Stem Cell‐Derived Neuroepithelial Cells

During neural development, cells generate and respond to mechanical forces within their microenvironment while they migrate and differentiate. These forces are central to the development of the human brain, as they are transduced into intracellular signals that regulate key neurobiological processes and may be involved in pathological processes. However, measurement of such forces in the nanonewton (nN) range within a three-dimensional (3D) microenvironment remains technically challenging and computationally intensive. Here, we present a solution to this challenge, based on elastomeric 3D microstructures fabricated via two-photon polymerization (2PP) and a tailored wet-etching process. The resulting free-standing beam architectures enable the quantification of nN forces exerted by pluripotent stem cell-derived neuroepithelial progenitor cells. We characterized the morphology of the cells and the forces they exerted using live-cell confocal imaging microscopy, scanning electron microscopy (SEM), supported by an in-house routine for intensity-based deflection measurements with sub-pixel resolution, and atomic force microscopy (AFM). Using this platform, we precisely quantified neuronal traction forces down to 1.2 nN from both healthy neural cells and those carrying a TSC2 gene mutation linked to tuberous sclerosis complex (TSC). The proposed elastomeric microenvironment paves the way for investigating cytoskeletal mechanics, neuromechanobiology in 3D, and for developing in vitro disease treatment models.