Dynamic cell patterning and photopolymerization with electric field modulation for constructing hierarchical tumor microenvironments

Engineered tumor models that replicate the hierarchical, heterogeneous microenvironment of in vivo tumors have shown huge potential in biomedical and clinical research. Tumor spheroids are widely used as in vitro models to investigate tumor pathophysiology. However, due to the complex cell-cell and cell-extracellular matrix (ECM) interactions in native tumor tissues, tumor spheroids alone often fail to replicate the intricate architecture and functionality of the tumor microenvironment (TME). Here, we propose a versatile strategy that assembles tumor spheroids while simultaneously constructing individualized ECM-mimicking environments, thereby more accurately replicating the native hierarchical TME. By applying a uniform electric field, the wettability of cell-laden hydrogel droplets is modulated, enabling their transport to predefined locations. Once positioned, a non-uniform electric field induces dielectrophoresis (DEP), guiding the cells to aggregate into tumor spheroids following predefined patterns. A digital micromirror device (DMD) then dynamically controls the shape and position of ultraviolet (UV) patterns, triggering photopolymerization of the hydrogel and precisely encapsulating the cell spheroids, thereby forming tumor models. In our experiment, breast cancer and liver cancer cells were aggregated to form tumor spheroids that maintained high cell viability, proliferative capacity, and morphological regularity, with the spheroid circularity reaching 0.84. Furthermore, when liver cancer spheroids were encapsulated in hydrogels containing endothelial cells, their invasiveness increased by approximately 77 %. We anticipate that our method will be capable of regenerating more complex tumor models with unprecedented possibilities for future drug discovery. STATEMENT OF SIGNIFICANCE: Reconstructing a hierarchical tumor microenvironment (TME) requires not only the formation of cell aggregates with natural intercellular connections but also the precise spatial organization of stromal cells and extracellular matrix components (ECM). However, achieving such dynamic cellular assembly and controllable heterogeneity during bioprinting remains challenging. Here, we present a multifunctional strategy that integrates dielectrophoretic droplet manipulation into a 3D bioprinting system to induce the in situ formation of compact, viable, and uniform tumor spheroids. Bioinks containing ECM components and stromal cells are then spatially patterned and photopolymerized with high precision to build customizable, biomimetic TMEs. This approach provides a versatile and controllable platform for drug screening, cancer research, and personalized medicine.