Co-culture of HepG2 and LX-2 cells enhances the compactness of 3D spheroids. (A) HepG2 cells, which formed loosely aggregated spheroids alone, were co-cultured as multilineage spheroids with LX-2 cells (forming round-shape spheroids quickly) at a 1:1 and 24:1 ratio for 24, 48, 72 and 96 h. Scale bars in bright-field pictures are 100 μm. (B) Spheroid volume was calculated measuring their long and short diameter by ZEN 2.3 Lite software (Zeiss) (n = 4). (C) Cellular ATP levels normalized to spheroids volumes remained stable throughout 4 days of culture (n = 3). (D) Apolipoprotein B (APOB) secretion levels in the media fractions measured by Western blotting are proportioned to the percentage of hepatocytes present in the spheroids (n = 5). Bars represent mean ± SD. P-values were calculated by Mann-Whitney non-parametric test, (* p < 0.05 vs. HepG2). APOB: Apolipoprotein B; AU: arbitrary unit.