Cellular Snowballing: Cell Adhesion and Migration Drive the Self‐Assembly of Cell‐Microgel Biohybrid Spheroids

ABSTRACT Creating three‐dimensional (3D) tissue models using cell spheroids that recapitulate the complicated structures and functions of human tissues is essential for advancing new approach methodologies used in drug testing/screening, disease modeling, and regenerative medicine. However, cell spheroids often have dense cellular structures and subsequently poor cell survival, primarily due to impaired oxygen and metabolite transport. To overcome these limitations, we develop biohybrid spheroids (BHS), self‐assembled living‐synthetic hybrid aggregates, using adherent cells as assembly engines and hydrogel microparticles (microgels) as extracellular matrix‐mimetic substrates. We show the revolving assembly of 3D BHS, driven by progressive cell migration and adhesion via culturing adherent mammalian cells and gelatin methacryloyl microgels, reminiscing a snowballing effect. The aggregation kinetics and terminal size of BHS are tailored by adjusting microgel size and cell‐to‐microgel ratio. Notably, microgels significantly larger than the cells yield porous, millimeter‐sized BHS, facilitating molecular diffusion and improving cell viability. Furthermore, transcriptional analyses show shifts in adhesion, angiogenesis, hypoxia, and proliferation programs in BHS compared with cell spheroids. An agent‐based model is developed to recapitulate the snowballing assembly in a geometrically unconstrained environment, providing fundamental insights into the assembly kinetics and the ultimate BHS size and pore features. BHS may open new opportunities for developing predictive and scalable technologies to self‐assemble large‐scale physiologically relevant tissue models in vitro, potentially transforming the biofabrication of microphysiological systems.