3C and 3B, respectively; Supporting Information Fig. 4). Quantification of platelet staining showed a statistically significant difference between seeded and
unseeded bioscaffolds find more (Fig. 4F), confirming the antithrombogenic function of the ECs in the seeded bioscaffolds. We performed series of coseeding experiments of human umbilical vein endothelial cells (hUVECs) and freshly isolated human fetal liver cells (hFLCs), using the methods developed for re-endothelialization of the bioscaffolds. An initial indication of successful cell seeding of the bioscaffold was apparent by a macroscopic change in the bioscaffold appearance from transparent white to opaque yellow 3-4 days after seeding (Fig. 5A). DNA extraction from a small sample of the seeded scaffold after 7 days revealed a 10-fold increase in DNA concentration (P = 0.0061) compared to an acellular scaffold (Supporting Information Fig. 1A). These DNA amounts correspond to approximately 37% of the DNA present
in fresh ferret liver. H&E staining showed dense cellularity throughout the whole seeded bioscaffolds (Supporting Information Fig. 3D). Proliferation was assessed by immunofluorescence staining for Ki67 and revealed a high number of positive cells detected throughout the bioscaffold (Fig. 6A). Accordingly, TUNEL staining showed a low number of apoptotic cells (Fig. 6B). Quantitative see more image analysis confirmed a 3× higher number of proliferating versus apoptotic cells Terminal deoxynucleotidyl transferase (Fig. 6C). Immunofluorescence staining showed hepatocytic lineage markers such as α-fetoprotein (Supporting Information Fig. 3E), CYP2A and CYP3A (Fig. 5B,C) were expressed by cells in the parenchyma. Double
immunostaining showed intense staining for cytokeratin 19 (CK19) in biliary tubular structures throughout the bioscaffold and clusters of albumin-expressing hepatocytes distributed in the parenchyma (Fig. 5D). There was very little coexpression of these markers by the same cells, suggesting that the bioscaffold contains discrete niches for bile duct and hepatocytes. A similar pattern was observed when sections were costained with CK19 (biliary epithelial cells) and CK18 (hepatocytes) (Supporting Information Fig. 3F). CK19 cells are seen in ductal structures and clusters of CK18 cells surrounding them and in the parenchyma. Some cells showed coexpression of CK19 and CK18, suggesting an immature hepatoblast phenotype.23 Endothelial cell markers such as Von Willebrand Factor (vWF) (Fig. 5E) and endothelial nitric oxide synthase (eNOS) (Fig. 5F) showed staining around the vascular structures of the bioscaffold.