# Whole Organ Engineering: Approaches, Challenges, and Future Directions
## 5 Recent Developments and Accomplishments
### Liver  
Bioengineering a whole liver has long been clinically appealing because of the liver's tremendous ability to regenerate and the therapeutic potential of transplanted livers, which are the current standard for treating end-stage liver disease. A focus of whole liver engineering has been research and development of an acellular tissue scaffold engineered from a whole liver, aiming to utilize the native structure to house parenchymal cells that will revive liver-specific functions. [PERSON] et al. published work demonstrating that perfusing rat livers with sodium dodecyl sulfate (SDS) solution followed by Triton X-100 solution through the hepatic portal vein resulted in the removal of cellular material, namely DNA, in the livers, while still retaining the native 3D ECM architecture and preserving the vascular architecture [40]. By recellularizing the acellular architecture with adult rat hepatocytes via their previously developed seeding protocol, [PERSON] and colleagues found that the hepatocytes remained viable for 5 days, with albumin and urea production levels during that time indicating that the cells were metabolically functional. The engineered livers were then heterotopically transplanted into rats, which demonstrated that engrafted hepatocytes were able to remain in their original attachment point and maintain their cellular morphology when exposed to blood flow from the host circulatory system, despite the resulting shear stress the circulation imparted on the cells. Furthermore, hepatic function such as secretion of albumin and urea as well as activity levels of CP450 were comparable to those of in vitro studies, which demonstrated the possibility for an engineered whole liver to function as a liver transplant in rats [40]. [PERSON] et al., also developed a bioengineered whole rat liver, in which human hepatic cells were successfully incorporated. Excised rat livers were perfusion decellularized using a solution of Triton X-100 mixed with ammonium hydroxide. The acellular liver scaffolds were then recellularized using human umbilical vein endothelial cells (HUVECs) and human fetal liver cells via perfusion of the preserved vascular network [63]. These rat livers cellularized with human cells were cultured in a bioreactor and displayed morphology and function resembling that of native liver tissue. The fetal liver cells and endothelial progenitor cells used were able to differentiate into mature endothelial and hepatic-specific cells. In 2015, [PERSON] and colleagues successfully conjugated anti-endothelial cell antibodies to the inner wall of blood vessels before recellularizing decellularized porcine livers, demonstrating that this approach could generate functionality within the liver vascular network [101]. This approach enabled endothelial cells to homogenously attach to both larger blood vessels as well as capillaries, significantly reducing the extent to which platelet adhesion occurred once in vitro blood perfusion was performed. After vascularization, the bioengineered porcine livers were capable of withstanding the shear stress arising from blood flow and successfully avoid thrombosis after implantation into pigs. In another study by [PERSON] et al. in 2016, instead of using anti-endothelial cell antibodies, a heparin-gelatin mixture was incorporated to improve the attachment efficiency of endothelial cells as well as mitigate thrombus formation [102]. Using this methodology, bioengineered livers that underwent vascular network reconstruction were also implanted and studied in porcine models. In 2011, [PERSON] et al., used perfusion with enzymatic and non-SDS detergent solutions along with mechanical stimulation to decellularize whole rat livers, with analysis of the decellularized whole liver matrices revealing that in addition to preserving the structure and vasculature of the liver ECM, their decellularization process maintained ECM composition and presence of growth factors, which can often be eliminated during decellularization [42].