Fabrication of Millimeter-Sized Three-Dimensional In Vitro Tissues with High Cellular Density and Hierarchical Vascular Networks

The bioengineering of three-dimensional (3D) tissues, such as spheroids and organoids, has transformed regenerative medicine, offering in vitro platforms for transplantation and drug screening. Typically, spheroids and organoids range from 50 to 200 μm in size. Larger 3D tissues (several millimeters) with high cellular density (≥10 8 cells/cm 3 ) enable the development of complex tissues containing vascular networks that closely mimic in vivo environments. However, fabricating such tissues is challenging due to oxygen and nutrient limitations. This study demonstrates the development of millimeter-sized 3D in vitro tissues with high cellular density through triculture with pericytes, 3D printing, and shaking culture. These three steps enabled the rapid formation of mature hierarchical vascular networks, integrating artificial channels and capillaries to supply oxygen and nutrients. Pericytes facilitated vasculogenesis and promoted anastomosis between artificial channels and capillaries. This work offers novel approaches to scale-up in vitro tissue models with vascularized networks for tissue engineering applications.