Mechanoscribed Xolography for Multiscale Mechanical Patterning of Cell-laden Hydrogels

Mechanotransduction is a key regulator of healthy and malignant tissue development. However, current mechanobiological insights are largely derived from cells cultured on two-dimensional substrates or in homogeneous biomaterials, which insufficiently capture the three-dimensional (3D) heterogeneity and dynamic nature of the native extracellular matrix. This gap persists due to a lack of manufacturing processes and analysis modalities to control and monitor material properties and their evolution in 3D.

Here, Mechanoscribed Xolography is introduced as a light sheet volumetric bioprinting process to rapidly fabricate 3D cell-laden hydrogels with spatially controlled mechanical heterogeneity across multiple length scales. Moreover, synchronizing in-situ optical microscopy with printing enables autonomous detection and spatial location-driven printing around cellular spheroids.

The refinement and spatiotemporal evolution of material properties are assessed with Brillouin microscopy, which verifies that the contrast of imprinted heterogeneity is preserved over three weeks. Remarkably, regional differences in cell behavior are attained within a one-step, one-material construct with elasticity of approximately 0.3 kPa–0.8 kPa, or 0.7 kPa–2.9 kPa with a crosslinking-enhancing additive. This foundational work on high-precision, rapid bioprinting surrounding cellular objects represents a unique tool to systematically advance experimentation of 3D mechanotransduction and prospectively engineer cell-instructive biomaterials for regenerative medicine.