Substrate Stiffness Modulates Cell-Network Topology in Human-Derived Neurons

Using confocal imaging and network topology analysis, we investigated how substrate stiffness affects adhesion and connectivity in hiPSC-derived neuronal cultures grown on polydimethylsiloxane (PDMS). We compared soft (∼12 kPa) and stiff (∼1.5 MPa) substrates with matched surface properties to isolate mechanical effects. Confocal analysis of NCAM expression revealed higher levels on soft PDMS at 10 DIV, indicating enhanced neuronal adhesion and outgrowth. Network topology analysis showed that only soft PDMS supported increased clustering, reduced path length, and higher small-worldness, reflecting more efficient connectivity. These results underscore the importance of substrate compliance in promoting neuronal development and inform scaffold design for neural engineering.