Cell Behavior within Nanogrooved Sandwich Culture Systems

Grooved topography and inherent cell contact guidance has shown promising results regarding cell proliferation, morphology, and lineage‐specific differentiation. Yet these approaches are limited to 2D applications. Sandwich‐culture conditions are developed to bridge the gap between 2D and 3D culture, enabling both ventral and dorsal cell surface stimulation. The effect of grooved surface topography is accessed on cell orientation and elongation in a highly controlled manner, with simultaneous and independent stimuli on two cell sides. Nanogrooved and non‐nanogrooved substrates are assembled into quasi‐3D systems with variable relative orientations. A plethora of sandwich‐culture conditions are created by seeding cells on lower, upper, or both substrates. Software image analysis demonstrates that F‐actin of cells acquires the orientation of the substrate on which cells are initially seeded, independently from the orientation of the second top substrate. Contrasting cell morphologies are observed, with a higher elongation for nanogrooved 2D substrates than nanogrooved sandwich‐culture conditions. Correlated with an increased pFAK activity and vinculin staining for sandwich‐culture conditions, these results point to an enhanced cell surface stimulation versus control conditions. The pivotal role of initial cell‐biomaterial contact on cellular alignment is highlighted, providing important insights for tissue engineering strategies aiming to guide cellular response through mechanotransduction approaches. Nanogrooved sandwich‐culture systems with relative orientation to assess cell response while stimulating cell dorsal and ventral receptors, featuring single and double‐seeded substrates. Cells acquire the orientation of the substrate on which they are initially seeded, independently of the orientation or existence of preseeded cells on opposing substrates, whereas cell elongation is effectively conditioned within sandwich‐culture.