Self‐Organized Vascularized Human Liver Spheroids: Serum‐Free Culture Conditions and Use as Tissue Building Blocks

ABSTRACT Engineering vascularized human liver tissue for in vitro and in vivo applications remains a major challenge. Here, we describe a scalable approach to generate human liver spheroids with self‐organized, lumen‐containing vascular networks and demonstrate their use as building blocks for fabricating vascularized single and multilayer tissue constructs. Spheroids were formed from HepaRG liver cells, human umbilical vein endothelial cells, and adipose tissue‐derived mesenchymal stem cells. Including the latter in specific ratios prevented a spatial segregation of hepatic and endothelial compartments, enabling endothelial network formation. We present two media for culturing these spheroids: a serum‐reduced medium and a defined serum‐free medium containing Gibco KnockOut Serum Replacement. These media supported the long‐term maintenance of hepatocytes in a metabolically active, relatively mature state, as well as the persistence of endothelial networks. Spheroid‐derived endothelial cells established anastomoses with external endothelial channels in microfluidic devices, and upon grafting into mouse liver tissue extended into the host parenchyma. Moreover, endothelial sprouts emerging from the spheroids formed inter‐spheroid connections within permissive hydrogels, a process that depended on the inter‐spheroid distance. Finally, we demonstrate the fabrication of planar tissue layers with vascularly interconnected spheroids and the creation of a macroscale tissue construct from several such layers.