Bile duct-like phenotype of hepatocytic aggregates after long-term culture. Spheroidal aggregates were cultured within a type I collagen gel matrix for 25 days (A–C) or 90 days (D–L) in the presence of insulin (10−7 mol/L) and EGF (10 ng/ml). A, D: H&E staining. B, C, E–G, I–L: Immunocytochemistry for albumin (B, E), CK19 (C, F), cytokeratin 7 (CK7) (G), laminin (I), type IV collagen (Col-IV) (J), Jagged1 (K), and Notch1 (L). H: Lectin cytochemistry for Dolichos biflorus agglutinin (DBA). Original magnifications, ×100.