Bioengineering liver tissue by repopulation of decellularised scaffolds

Table 3 Liver decellularisation recellularisation studies

Species	Decellularisation method	Recellularisation cell type and route	Outcome	Ref.
Female Lewis rats	SDS + Triton X-100	(1) Primary rat hepatocytes via the Portal vein; and (2) Rat cardiac microvascular endothelial cells via portal vein	(1) Demonstrated Successful decellularization/Recellularization with cell viability and function; (2) Demonstrated the feasibility of transplanting these recellularised liver grafts in vivo with minimal ischemic damage; and (3) The recellularised graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro.	Uygun et al[86]
Fisher 344 rats	Triton X-100 + SDS	Rat liver progenitor cell line WB344 through the inferior vena cava	(1) Perfusion with 0.1% SDS for 1 hour completely cleared all DNA; and (2) Supplementation of all perfusion solutions with antibiotics/antimycotics prevented microbial growth, and the IDL could be stored at 4°C for several weeks.	Shupe et al[156]
Male Sprague Dawley rats	Trypsin + EGTA + Triton X-100	Primary mice hepatocytes via: (1) Direct parenchymal injection; (2) Continuous perfusion via the portal vein; and (3) Multistep infusion via the portal vein	Systematic comparison of three different reseeding methods showed that a multistep strategy provides the greatest seeding efficiency and the presence of functional hepatocytes.	Soto-Gutierrez et al[164]
Male Lewis rats	SDS + Triton X-100	Primary rat hepatocytes via the portal vein (from spheroid culture)	(1) Layer-by-layer heparin deposition was used to avoid thrombosis, followed by repopulation of hepatocytes, and successfully implanted as a TEL into the portal system; (2) Treatment of extended hepatectomized rats with a TEL improved liver function and prolonged survival; mean lifespan was extended from 16 to 72 h; and (3) At 72 h post operation, the TEL sustained functional and viable hepatocytes.	Bao et al[174]
Ferret	Distilled water + Triton X-100 + ammonium hydroxide	Human foetal liver cells + human umbilical vein endothelial cells co-infusion via the portal vein	Demonstrated delivery of cells to different compartments of the liver tissue via different pathways EC delivered through the vena cava selectively seeded larger and smaller blood vessels up to the pericentral area of the liver lobule and cells seeded through the portal vein reached predominantly the periportal area of the liver lobule.	Baptista et al[90]
Adult male Sprague–Dawley rats	SDS or Triton X-100 + sodium hydroxide	Primary rat hepatocytes via the portal vein	Decellularised scaffolds constructed by perfusion of Triton X-100 were of superior quality and can provide a more effective and ideal scaffold for tissue engineering and regenerative medicine.	Ren et al[161]
Porcine	SDS + DNase	Porcine hepatocytes via the portal VEIN	Demonstrated a protocol to decellularise rapidly a full-size porcine liver with small detergent volumes within 24 h.	Bühler et al[153]
Human	Distilled water + SDS + Triton X-100	Human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2) via suspension	Decellularised human liver cubic scaffolds were repopulated for up to 21 d using human cell lines with excellent viability, motility and proliferation and remodelling of the extracellular matrix.	Mazza et al[154]
Piglet	Triton X-100 + ammonium hydroxide	Murine endothelial cells (MS1) with combination of static and perfusion techniques (via the portal vein)	(1) Developed an effective method for re-establishing the vascular network within decellularised liver scaffolds by conjugating anti-endothelial cell antibodies to maximize coverage of the vessel walls with endothelial cells; (2) This procedure resulted in uniform endothelial attachment throughout the liver vasculature extending to the capillary bed of the liver scaffold and greatly reduced platelet adhesion upon blood perfusion in vitro; and (3) The reendothelialized livers, when transplanted to recipient pigs, were able to withstand physiological blood flow and maintained for up to 24 h	Ko et al[89]
Porcine	SDS + Triton X-100	Rat primary hepatocytes and human umbilical vein endothelial cells (cells cultured in scaffolds, but not in a perfusion circuit)	(1) The heparinized scaffolds showed improved anticoagulation and cytocompatibility compared to the control scaffold both in vitro and in vivo test; and (2) The layer-by-layer technique showed that heparinisation did not interfere with hepatocyte or endothelial cell repopulation.	Bao et al[176]
Porcine	SDS	Human EA.hy926 endothelial cells and HepG2 hepatic carcinoma cells via the portal vein	(1) The study demonstrated, exposing scaffold to heparin-gelatin mixture improved endothelial cell ability to migrate and cover vessel discs, perhaps by exploiting gelatin’s multiple integrin binding sites which facilitate endothelial cell binding; and (2) Scaffolds repopulated with Hep G2 hepatocytes and endothelial cells after heparin gelatin coating showed improved ex vivo blood perfusion, in comparison to uncoated scaffolds.	Hussein et al[87]
Male Lewis rats	Trypsin + EGTA + Triton X-100	Primary rat hepatocytes via the bile duct and the portal vein	The study results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.	Ogiso et al[173]
Male Lewis rats	Trypsin + EGTA + Triton X-100	(1) Primary rat hepatocytes via the Bile duct; and (2) LSECs via the portal vein	(1) Hepatocytes co-seeded with LSECs retained their function compared with those seeded alone; (2) LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity; and (3) Successfully achieved continuous blood flow into the vascularized liver graft by extracorporeal perfusion for at least 8 hours	Kojima et al[172]
Female Lewis rats	SDS + Triton X-100	Human EA.hy926 endothelial cells via the portal vein	(1) Coupled the cell-binding domain REDV to the vasculature of decellularised rat livers; and (2) REDV coupling increased cell attachment, spreading and proliferation of endothelial cells within the scaffold resulting in uniform endothelial lining of the vasculature, and a reduction in platelet adhesion and activation	Devalliere et al[88]
Female Lewis rat	SDS	(1) Rat cholangiocytes via the common bile duct; and (2) Rat hepatocytes via the portal vein	(1) Demonstrated for the first time, whole liver grafts co-populated with hepatocytes and cholangiocyte; (2) Cholangiocytes formed duct-like structures, with the viable hepatocyte mass residing in the parenchymal space, in an arrangement highly comparable to the native tissue; and (3) Both albumin and urea assay results confirmed hepatocyte functionality and the gene expression analysis of cholangiocytes in recellularised liver grafts indicated viability and sustained gene expression of functional proteins.	Chen et al[177]
Adult Sprague–Dawley rats	Triton X-100 + NH4OH	Rat sinusoidal endothelial cells were perfused via the Portal vein in either RPMI media or in 5% gelatin hydrogel solution	(1) Used immortalized endothelial cells to repopulate decellularised rat liver scaffolds; (2) Gelatin hydrogels-based perfusion significantly increased the number of cells that were retained in the scaffolds; and (3) The Doppler ultrasound detected active blood flows within the re-endothelialised liver scaffolds 8 d post heterotopic transplantation.	Meng et al[190]
Male Lewis rats	Trypsin/EGTA solution + Triton X-100/EGTA	Human induced pluripotent stem cells derived hepatocyte-like cells via bile duct	(1) The first study to generate a recellularised liver model with human hepatic function using human induced pluripotent stem cells; and (2) This result suggested that the BD was an appropriate recellularization pathway regardless of the hepatocyte type.	Minami et al[250]
Porcine	SDS + Triton X-100	Human umbilical vein endothelial cells via the superior vena cava followed by via the portal vein	Decellularised whole porcine livers revascularized with human umbilical endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleen has been removed sustained perfusion for up to 20 d.	Shaheen et al[191]
Porcine	Triton X-100 + SDS	(1) Human umbilical vein endothelial cells via the vena cava and the portal vein; and (2) Porcine hepatocytes via the bile duct	(1) Co-seeded primary porcine hepatocytes after human umbilical vein endothelial cell reendothelialization; and (2) Repopulated scaffolds were implanted heterotopically in a pig model and produced improved biochemical function in an acute liver failure model.	Anderson et al[175]
Female Sprague-Dawley rats	SDS + DNase	Human umbilical vein endothelial cells via the Portal vein	(1) Used aptamers (short, single-stranded DNA or RNA molecules that selectively bind to specific targets) with CD31 specificity; and (2) Aptamer coated scaffolds showed higher endothelial cell coverage, enabled perfusion with blood for 2 h with reduced platelet adhesion ex vivo, and restored liver function in a hepatic fibrosis rat model.	Kim et al[192]

TEL: Tissue-engineered liver; SDS: Sodium dodecyl sulphate; LSECs: Liver sinusoidal endothelial cells; REDV: Arg GluAsp Val.