Culture Architecture Shapes Distinct Functional and Transcriptional Profiles of AML12 Hepatocytes Across 2D and 3D Configurations

Background/Objectives: Simple, reproducible in vitro liver models are increasingly important for early screening strategies aligned with the 3Rs principles. However, three-dimensional culture effects depend strongly on spatial organization, and greater structural complexity does not nec-essarily improve functional performance. This study compared five AML12 culture configura-tions to evaluate their effects on hepatocyte-associated characteristics. Methods: AML12 cells were cultured for 7 days under matched conditions using conventional tissue-culture plastic (2D-TC), two-dimensional collagen-supported culture (2D-Coll), three-dimensional culture on a collagen gel (3D-Coll), collagen sandwich culture (3D-SW), and collagen-supported spheroid cul-ture (3D-Sph). Cellular organization, cell abundance and size, metabolic and biochemical param-eters, and transcriptional markers were evaluated using four biological replicates for gene-expression analyses and six for biochemical measurements. Multivariate analyses were used to characterize transcriptional patterns. Results: Cell number remained comparable across con-figurations, whereas cell size, metabolic activity, biochemical endpoints, and transcriptional pro-files differed. The collagen sandwich produced the most consistent hepatocyte-associated profile, with increased Alb, Hnf4a, Ttr, and Trf expression and the highest urea production. Spheroids showed the highest cell-normalized resazurin activity and multicellular aggregation, while 2D collagen exhibited a stronger stress-associated profile. PERMANOVA confirmed an overall effect of culture configuration on transcriptional profiles (R² = 0.442, p < 0.001). Conclusions: AML12 responses depended on culture configuration rather than following a simple 2D-versus-3D pattern, highlighting the importance of spatial organization when selecting in vitro liver models.