Kaysan, L. | bioRxiv Science

bioRxiv2025

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 GibcoTM 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/684548v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1b8c8a2org.highwire.dtl.DTLVardef@7c0ed2org.highwire.dtl.DTLVardef@b10886org.highwire.dtl.DTLVardef@a6ab4f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Human cerebral organoids model tumor infiltration and migration supported by astrocytes in an autologous setting

Efforts to achieve precise and efficient tumor targeting of highly malignant brain tumors are constrained by the dearth of appropriate models to study the effects and potential side effects of radiation, chemotherapy, and immunotherapy on the most complex human organ, the brain. We established a cerebral organoid model of brain tumorigenesis in an autologous setting by overexpressing c-MYC as one of the most common oncogenes in brain tumors. GFP+/c-MYChigh cells were isolated from tumor organoids and used in two different culture approaches: assembloids comprising of a normal cerebral organoid with a GFP+/c-MYChigh tumor sphere and co-culture of cerebral organoid slices at air-liquid interface with GFP+/c-MYChigh cells. GFP+/c-MYChigh cells used in both approaches exhibited tumor-like properties, including overexpression of the c-MYC oncogene, high proliferative and invasive potential, and an immature phenotype as evidenced by increased expression of Ki-67, VIM, and CD133. Organoids and organoid slices served as suitable scaffolds for infiltrating tumor-like cells. Using our highly reproducible and powerful model system that allows long-term culture, we demonstrated that the migratory and infiltrative potential of tumor-like cells is shaped by the environment in which glia cells provide support to tumor-like cells.

cell biology↗