A 3D co-culture strategy combining fetal liver bud-derived cells and adipose-derived mesenchymal stromal cells for spheroid formation and decellularized liver scaffold colonization

Engineering liver-like constructs remains challenging because hepatic cell function depends on coordinated interactions between parenchymal progenitors, supportive stromal cells, and the highly organized extracellular matrix (ECM) microenvironment of the liver. Although three-dimensional culture can enhance cell–cell interactions, reproducing the structural and biochemical complexity of native hepatic tissue remains difficult. Decellularized liver scaffolds provide an organ-specific ECM while preserving relevant tissue architecture, whereas mesenchymal stromal cells may provide complementary cellular support to developing hepatic populations. Here, we investigated whether combining E12.5 fetal liver bud-derived cells (LBCs) with adipose-derived mesenchymal stromal cells (AD-MSCs) could support 3D spheroid organization and subsequent colonization of decellularized rat liver scaffolds. LBCs isolated at different developmental stages exhibited distinct proliferative and functional profiles, with E12.5 cells showing the highest proliferative capacity and prominent progenitor-associated features while retaining measurable hepatic-associated function. Rat livers were decellularized by vascular perfusion, resulting in extensive cellular removal while preserving a porous and fibrillar ECM containing glycosaminoglycan-associated components, collagens I and III, biglycan, decorin, fibronectin, and vitronectin. Three-dimensional vascular reconstruction further demonstrated preservation of an extensive branching vascular network. E12.5 LBCs and AD-MSCs spontaneously assembled into compact and viable spheroids, and their co-culture maintained expression of AFP and albumin together with stromal-associated markers. Functionally, co-culture increased albumin release but reduced urea levels compared with LBCs spheroids alone, indicating differential modulation rather than uniform enhancement of hepatic-associated function. Finally, LBCs/AD-MSC co-cultures were introduced onto decellularized liver scaffolds under dynamic rotary culture. After 7 days, cells showed extensive attachment and interaction with ECM fibers, while nuclear staining demonstrated cellular distribution across different scaffold regions and penetration beyond the external surface. Together, these findings establish an experimental platform integrating fetal liver progenitor/stromal co-culture, scaffold-free 3D organization, and organ-derived ECM, providing a basis for further studies addressing cell-specific fate, spatial organization, vascular recellularization, and functional maturation of engineered liver constructs.