Multidimensional culture system for SH-SY5Y cells on microwell patterns regulated by the ROCK signaling pathway

This study presents the fabrication of polylactic acid microwell patterns using a lithography-based replica molding method for the development of three-dimensional (3D) neural cell-based assays. SH-SY5Y human neuroblastoma cells interfaced with these patterns, and cell growth and morphological characteristics were evaluated with and without an inhibitor of the rho-associated coiled-coil kinase (ROCK) signaling pathway. The results revealed that the culture systems of SH-SY5Y cells on the microwell patterns could be categorized as two-dimensional (2D), 3D, and near-two-dimensional (N2D), according to their location within a dynamic multidimensional culture system. Furthermore, the geometric features of the patterns significantly impact the efficiency of the conversion model, which was characterized by the proliferation and percentage of cells in different culture systems. The rearrangement of the microfilament cytoskeleton and morphological spreading of cells on the patterns were suppressed by the downregulation of the ROCK signaling pathway. The inhibition of the ROCK signaling pathway had a time-dependent effect on the proliferation and migration of cells on the patterned substrate, where topographical cues, cell morphology, and density played pivotal roles. Consequently, the proposed system serves as a practical model for studying the functional behaviors of neural cells by dynamically assembling multidimensional culture systems on the same platform, facilitating the development of cell-based assays.

Graphical abstract

ROCK-myosin II signaling pathway on the morphology and actin cytoskeleton of SH-SY5Y cells. Immunofluorescent staining for F-actin of FS-2D cells growing on flat substrates and N2D cells or 3D cells cultured on 100–20 μm pattern for 24 h with or without Y-27632 (d–f) and blebbistatin (g–i) treatment or without any treatment (a–c). Scale bar: 20 μm