Spatial Control of Light-Responsive Proteins and Optogenetics Within Hydrogels via Volumetric Bioprinting.

Spatiotemporal control over cell fate and behavior within bioprinted constructs remains a key challenge in tissue engineering. Optogenetics offers versatile potential for non-invasive regulation of biological processes. Yet, its integration within large-scale, cell-laden bioprinted materials is still limited, especially considering the spatial constraints of existing light delivery methods. In this study, we introduce a novel approach that repurposes tomographic volumetric bioprinting to enable post-printing stimulation of photosensitive protein-switches and optogenetic circuits in cells deep within hydrogel constructs. By converging different bioprinting approaches, computer vision, context-aware model generation, and synthetic biology and cell engineering, we demonstrated selective activation of a fluorescent, light-responsive protein probe within multi-material centimeter-scale constructs. Moreover, leveraging a multi-wavelength volumetric bioprinter, we further demonstrate this concept by selectively stimulating cells expressing a near-infrared optogenetic system that triggers gene expression and the induction of pancreas-specific transcription factors. The described methods provide platforms for remote, repeatable, and localized control of biological events in volumetric constructs, opening new possibilities for advanced tissue models, and dynamic tuning of cell-mediated protein production in engineered living systems.