Rapid Generation of Fusable Cell Beads for Multi‐Scale Human Living Materials Assembly

Three-dimensional self-assembled cellular aggregates, such as spheroids, provide unique building blocks for bottom-up tissue engineering and in vitro disease modeling. Nevertheless, traditional spheroid production methods require prolonged cell aggregation times and are highly dependent on cell type, requiring frequent optimization steps. Additionally, spheroids' size is dependent on their cell density, preventing a control over their final volume. Herein, a methodology combining metabolic glycoengineering and click chemistry with superhydrophobic surfaces is described to rapidly create spherically structured living bead units, that can surpass the fabrication constraints of conventional spheroids. Compared to spheroids produced in low attachment settings, the living beads comprising various cell types (i.e., stem, endothelial, and cancer cells) are rapidly produced and demonstrate enhanced cell viability and cell spreading over 14 days, while maintaining principal spheroid characteristics, namely the fusion into multi-scale living materials and cellular migration capabilities. In addition, this methodology enables the production of living beads with controlled size, independently of cell density, overcoming a key limitation of current spheroid production methods. The enhanced reproducibility, reduced cell assembly time, and improved handling make these spherically structured living beads a valuable alternative, with broad application in bottom-up tissue engineering approaches and disease modeling applications.