Differences in albumin and apolipoproteins secretion between monolayer and spheroid HepG2 cells. (A) Representative appearance of HepG2 spheroids on Day 11. HepG2 spheroids were fixed with 10% formalin solution, and examined with confocal microscopy. Briefly, from a single HepG2 cell, cells proliferate and form a spheroidal cell-cluster in an aliginate bead. (B-E) Time-course changes in the secretion of albumin (B), apoA-I (C), apoE (D), and apoB (E) from S-Hep cells. S-Hep (spheroid HepG2 cells) were prepared and grown in alginate beads as described in Materials and Methods, and the protein levels in the media on different time points after the beginning of the culture were measured. X axes represent the days after the beginning of S-Hep culture. Data are mean ± SEM (n = 4). *: P < 0.01 compared with M-Hep; †: P < 0.03 compared with M-Hep; ‡: P < 0.01 compared with M-Hep and P < 0.03 with S-Hep on the other time points.