(A) HepG2 cells were seeded into 96-well plates at 250 and 500 cells/well. After 5 days of culture, cells were refed with fresh media and subsequently cultured for 24 h. Media and cells were then separately collected and relative amounts of albumin and viable cell signals were respectively measured as described in the ‘Materials and Methods’ section. Error bars indicate ± S.E.M. (n = 4). **P<0.01, compared with 2D cultures at each cell count. (B) HepaRG cells were seeded into 96-well plates at 72000 cells/well (2D) or 24000 cells/well (3D) and cultured according to the procedures listed in the ‘Materials and Methods’ section. After 6 days of culture, cells were refed with fresh media and processed in the same manner as (A). Error bars indicate ± S.E.M. (n = 6). **P<0.01, compared with 2D cultures.