Mechanical regulation of N2A neural cell behavior by microtissue densification

Tissue densification, as a hallmark of development, injury, and fibrosis, alters the mechanical and structural properties of the extracellular matrix (ECM). However, its specific effects on neural cell behavior remain poorly understood. To address this, we developed a 3D in vitro microtissue model composed of collagen and Matrigel, incorporating co-cultures of N2A neuroblastoma and 3T3 fibroblast cells. By modulating fibroblast-driven contraction through scaffold-guided constraint, we generated microtissues with distinct levels of densification with Young's moduli ranging from approximately 0.5 to 1 kPa. Our results demonstrated that increased microtissue densification significantly enhanced N2A migration and aggregate formation, indicating that mechanical compaction facilitates neuronal clustering. Furthermore, higher densification promoted N2A cell proliferation, while apoptosis remained at relatively low baseline levels, suggesting that dense environments support cell expansion without affecting overall viability. Additionally, higher densification suppressed the proportion of neurite-bearing cells without affecting neurite length, implying impaired initiation of neuronal differentiation but not morphological maturation. Together, these findings reveal that ECM densification serves as a critical mechanical cue shaping multiple aspects of neural cell behavior. Compared to conventional hydrogel systems, our collagen-based microtissue model provides a more physiologically relevant in vitro platform for studying neurodevelopmental mechanobiology and guiding biomaterial design for neural tissue engineering.