Effects of fluid flow rate on cellular organization. (A) Representative images of albumin staining of HepG2 cells (green) and eNOS staining of MS1 cells (red) from days 1 (top) and 7 (bottom), at the indicated flow rates (white arrows point to vascular structures). (B) Coimmunostaining of cells expressing albumin, eNOS and Ki-67 (inset). (C) Analysis of cell types (MS1+HepG2, MS1 only, and no cells) covering the vascular structures at 3, 6, 9, and 12 mL/min, 1 and 7 days postcell seeding. (D) HepG2 cluster size at 1 and 7 days postcell seeding. (E) An animation of cellular organization in the liver scaffold during perfusion seeding. Results presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 100 μm (A), 50 μm (B). eNOS, endothelial nitric oxide synthase.