DLM retains intact lobular structure and vascular bed. (a) Representative photograph of decellularized left lateral and median lobes of rat liver, with the vascular tree visible. (b) The vascular tree, after perfusion with Allura Red AC dye. (c,d) Corrosion cast model of left lobe of a normal liver (c) and the DLM (d), with portal (red) and venous (blue) vasculature. (e–g) SEM images of a vessel (e), a section featuring bile duct–like small vessels (arrows) (f), extracellular matrix within the parenchyma (g), with hepatocyte-size free spaces. Scale bars: 10 mm (a,b), 5 mm (c,d) and 20 μm (e–g).

Hepatic function of the recellularized liver graft in vitro. (a,b) Albumin synthesis (a) and urea secretion (b) in comparison to static sandwich culture (P values are 0.0176 for albumin and 0.0017 for urea, by Friedman’s test). (c) Gene expression analysis of hepatocytes in the recellularized liver graft (2 d) compared to normal liver, fresh hepatocytes and sandwich culture hepatocytes (2 d) for phase 1 and phase 2 drug metabolism enzymes. (d) Scatter plot comparing gene expression of phase 1 and phase 2 drug metabolism enzymes at 2-d recellularized liver graft and cultured hepatocytes (P = 0.0499 by Friedman’s test). (e–j) Normalized gene expression of Cyp2c11 (e), Gstm2 (f), Ugt1a1 (g), Cyp1a1 (h), Adh1 (i) and Cyp3a18 (j). All error bars represent s.e.m. (n = 3).

Decellularization of ischemic rat livers. (a–e) Representative images of ischemic rat livers during decellularization process at 0 h (a), 18 h (b), 48 h (c), 52 h (d) and 72 h (e). The livers were perfused through the portal vein using SDS as described in the Online Methods. (f) Comparison of normal liver (top) and DLM (bottom). Left to right: H&E, collagen I (red), collagen IV (red), fibronectin (red) and laminin (red) staining. Sections were counterstained with DAPI (blue). Scale bars: 10 mm (a–e) and 100 μm (f).

Repopulation of rat DLM with adult rat hepatocytes. (a) Recellularization scheme of the DLM. (b,c) Decellularized whole liver matrix (b) and same liver after recellularization with about 50 million hepatocytes (c). (d) TUNEL staining of recellularized liver grafts. Left to right: recellularized graft at 48 h in culture (arrows indicate positive cells), fresh isolated liver (negative control), DNase-treated normal liver (positive control). Insets show Hoechst 33258 counterstain of the same sections. (e) TUNEL-positive cell percentage in recellularized liver grafts as a function of perfusion-culture time. (f) LDH release from recellularized livers during perfusion culture (P = 0.0455 by Friedman’s test). (g) SEM micrographs of recellularized liver graft after 2 d in culture. (h) Immunohistochemical staining of the recellularized liver graft (bottom) in comparison to normal liver (top); left to right: H&E, albumin (red), glucose 6-phosphatase (red) and Ugt1a (green). Sections were counterstained with Hoechst 33258 (blue). Scale bars: 20 mm (b,c), 200 μm (d) and 100 μm (h). All error bars represent s.e.m. (n = 6).

Transplantation of the recellularized liver graft. Decellularized and recellularized rat liver was transplanted as auxiliary heterotopic graft with portal vein arterialization, and graft viability was determined 8 h after transplantation. (a) Representative images of graft transplantation; top, left to right: transplant site, recellularized graft at the transplant site, transplanted graft before blood reperfusion, the graft right after declamping the renal artery; bottom, left to right: transplanted graft 1 min, 2 min and 4 min after declamping of the renal artery, and auxiliary graft in contrast with the native liver. (b) Immunohistochemical staining of recellularized auxiliary graft 8 h after transplantation (bottom) compared to native liver (top); left to right: H&E, albumin (red), glucose 6-phosphatase (red), Ugt1a (green). Sections were counterstained with Hoechst 33258 (blue). Scale bars: 10 mm (a) and 100 μm (b).