Fabrication of heterocellular spheroids with controllable core-shell structure using inertial focusing effect for scaffold-free 3D cell culture models

Abstract Three-dimensional (3D) cell culture models capable of emulating the biological functions of natural tissues are pivotal in tissue engineering and regenerative medicine. Despite progress, the fabrication of in vitro heterocellular models that mimic the intricate structures of natural tissues remains a significant challenge. In this study, we introduce a novel, scaffold-free approach leveraging the inertial focusing effect in rotating hanging droplets for the reliable production of heterocellular spheroids with controllable core-shell structures. Our method offers precise control over the core-shell spheroid’s size and geometry by adjusting the cell suspension density and droplet morphology. We successfully applied this technique to create hair follicle organoids, integrating dermal papilla cells within the core and epidermal cells in the shell, thereby achieving markedly enhanced hair inducibility compared to mixed-structure models. Furthermore, we have developed melanoma tumor spheroids that accurately mimic the dynamic interactions between tumor and stromal cells, showing increased invasion capabilities and altered expressions of cellular adhesion molecules and proteolytic enzymes. These findings underscore the critical role of cellular spatial organization in replicating tissue functionality in vitro. Our method represents a significant advancement towards generating heterocellular spheroids with well-defined architectures, offering broad implications for biological research and applications in tissue engineering.