The present work explores cell cultivation in macroporous alginate scaffolds as a means to reproduce hepatocyte terminal differentiation in vitro. Newborn rat liver cell isolates, consisting of proliferating hepatocytes and progenitors, were seeded at high cell density of 125 x 10⁶/cm³ within the scaffold and then cultivated for 6 wk in chemically defined medium. Within 3 days, the alginate-seeded cells expressed genes for mature liver enzymes, such as trypthophan oxygenase, secreted a high level of albumin, and performed phase I drug metabolism. The cells formed compacted spheroids, establishing homotypic and heterotypic cell-to-cell interactions. By 6 wk, the spheroids developed into organoids, with an external mature hepatocyte layer covered by a laminin layer encasing inner vimentin-positive cells within a laminin-rich matrix also containing collagen. The hepatocytes presented a distinct apical surface between adjacent cells and a basolateral surface with microvilli facing extracellular matrix deposits. By contrast, viable adherent cells within collagen scaffolds presenting the identical porous structure did not express adult liver enzymes or secrete albumin after 6 wk. This study thus illustrates the benefits of cell cultivation in macroporous alginate scaffolds as an effective promoter for the maturation of newborn liver cells into functional hepatic tissue, capable of maintaining prolonged hepatocellular functions.—Dvir-Ginzberg, M., Elkayam, T., Cohen, S. Induced differentiation and maturation of newborn liver cells into functional hepatic tissue in macroporous alginate scaffolds.