Microfabrication of Substrates with Microscale Stiffness Gradients to Guide Bone Marrow Stromal Cell Migration

Understanding how cells respond to mechanical signals is crucial for elucidating the mechanisms involved in tissue development and disease progression. However, existing in vitro cell culture substrates often fail to replicate the physiological stiffness gradients at the cellular scale while also eliminating confounding topographical cues. In this study, we present a decoupled stiffness model utilizing a bilayer polydimethylsiloxane (PDMS) substrate. This substrate consists of a soft, flat top film suspended over a rigid underlayer featuring ridge-and-groove microstructures. The top layer effectively transmits stiffness variations in the underlying structure while maintaining uniform topography and chemistry at the cell contact surface. Stiffness modulation is achieved by varying the width of the alternating ridges and grooves, which are spaced equally. Scanning electron microscopy confirmed the flat surface morphology and consistent contact topography. Atomic force microscopy demonstrated that stiffness variations were dependent on the microstructure: for 20 μm patterns, the elastic moduli were approximately 950 kPa for ridges and 850 kPa for grooves; for 50 μm patterns, these values were around 1070 kPa and 950 kPa, respectively. Mouse primary bone marrow cells adhered well and spread on the substrate, showing a preference for nuclear localization toward the stiffer ridge regions in the 50 µm pattern (61.49%, p < 0.05), thereby confirming effective cellular perception of the mechanical gradient. In summary, this protocol offers a reproducible method to construct a cell culture substrate with microscale stiffness gradients, minimizing the chemical or topography interference, enabling investigations into cell mechanotransduction.