FUS-pen: focused ultrasound-mediated micro-sculpting of label-free cells to create functional co-culture microenvironments.

Precise spatial organization of cells and microparticles is essential for engineering physiologically relevant tissues and in vitro disease models, yet current bioink-based bioprinting approaches still face limitations regarding cell density dilution and crosslinking-induced cellular stress. Here we introduce the FUS-pen system, a template-free, focused ultrasound platform that enables non-contact, flexible and cross-scale manipulation of microparticles and cells with demonstrated biocompatibility. Mechanistic investigations reveal a cooperative interaction where acoustic streaming drives long-range transport and localized acoustic radiation forces achieve near-focus confinement of microparticles, while boundary reflections establish standing-wave-like fields that yield distinct, frequency-dependent swarm dimensions. By optimizing GelMA hydrogel substrates for reliable cell pattern retention and integrating poly-D-lysine (PDL) modification to facilitate electrostatically driven cell interface interactions, we achieved a quantified sub-millimeter feature resolution (∼293 μm line width), consistent geometric spot reproducibility and high cell viability (>94%). As a proof of concept, a compartmentalized tumor-endothelial co-culture layout was successfully established using breast cancer (MCF-7) cells and endothelial (EA.hy926) cells, where endothelial cells exhibited active proliferation and morphological remodeling into capillary-like networks, demonstrating the platform's utility for constructing complex multicellular models. Together, these results establish the FUS-pen as an agile tool for constructing organized in vitro microenvironments with broad implications for disease modeling and drug screening.