Self-organized vascularized human liver spheroids: Serum-free culture conditions and use as tissue building blocks

Engineering vascularized human liver tissue for in vitro models 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 tissue layers. Spheroids were formed from HepaRG liver cells, human umbilical vein endothelial cells (HUVECs), and adipose tissue-derived mesenchymal stem cells (MSCs). The inclusion of MSCs prevented spatial segregation of hepatic and endothelial compartments and enabled endothelial network formation. We present two media that are suitable for culturing these spheroids: a serum-reduced medium and a defined serum-free medium containing Gibco ^TM 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. Endothelial cell identity and organization were confirmed by VE-cadherin and ICAM-2 immunostaining and by transmission electron microscopy, which revealed adherens junctions and luminal morphologies consistent with a capillary-like organization. Spheroid-derived HUVECs established anastomoses with external endothelial channels in microfluidic devices. Moreover, endothelial sprouts emerging from the spheroids formed inter-spheroid connections within permissive hydrogels (fibrin or collagen–methylcellulose), a process that depended on the inter-spheroid distance. Finally, we demonstrate the fabrication of planar tissue layers with vascularly interconnected spheroids. Together, we identify key conditions, including cellular ratios, medium formulations, biomaterials, and spatial design criteria that enable the generation and assembly of vascularized liver spheroids as scalable tissue building blocks for tissue engineering applications.