The present study describes a minimally disruptive whole-organ decellularization protocol that produces a three-dimensional liver matrix suitable for supporting functional hepatocytes, and a comprehensive characterization of the decellularized liver matrix shows maintenance of the hepatic matrix ultrastructure and preservation of representative growth factors such as bFGF and HGF. Importantly, a comparison of three methods for reintroducing cells to the matrix identified a multistep perfusion technique that results in 90% grafting efficiency.
The results of the present study not only confirm the concept of whole-organ decellularization, as previously shown,5–7,13,21
but also provide new findings not reported in previous studies that will be important for clinical translation of this regenerative medicine approach for liver replacement. First, thorough decellularization can be accomplished in ~48
h without the use of harsh detergents such as sodium dodecyl sulfate. Second, growth factor content can be preserved within the matrix scaffold at 30%–50% of the native tissue. Third, decellularization was quantified by stringent and conservative criterion showing absolute amounts of residual DNA and not just percent decrease, and that any residual DNA consisted only of lengths no >200
bp; stated differently, very thorough decellularization was accomplished. As shown in previous studies, DNA fragments with <300
bp in length have shown no significant inflammatory reactions in several commercially available ECM products and ECM scaffold materials produced in the laboratory.19
Fourth, the biliary network was shown to be intact, along with the vascular structures. Fifth, Glisson's capsule was shown to be intact, an important feature because of anticipated future clinical applications in which hemorrhage after connection to an in vivo
vascular supply would be problematic. Finally, systematic comparison of three different reseeding methods showed that a multistep strategy provides the greatest seeding efficiency and the presence of functional hepatocytes.
One of the main limiting factors in the advancement of liver tissue engineering is the lack of an ideal transplantable scaffold that has all the necessary microstructural and extracellular cues for cell attachment, differentiation, functioning, and vascularization for oxygen and nutrient transport.14,22,23
Thus, the ability to create a transplantable liver scaffold is an important advancement in proof of concept. Whole-organ decellularization has been recently reported for different organs, including liver4–7,24
; however, in vivo
implantation has been limited to a few hours due to problems such as hemorrhage and thrombosis. The method for whole-liver decellularization reported herein directly addresses some of the issue that will determine ultimate clinical success.
The ECM in the developing liver has dynamic structural and functional features that mature with age and which directly affect the fate and gene expression of hepatoblasts.25
The various growth factors and chemo attractants are immobilized by ECM components through disulfide bonds.26,27
Thus, the well-defined ECM provided in the adult liver could potentially facilitate and guide the developmental pathway for full hepatic maturation of fetal liver cells or embryonic and/or induced pluripotent stem cells. Organ decellularization could potentially become a tool for stem cell differentiation and maturation to eventually engineer autologous liver grafts for transplantation.28
Studies with decellularized lung show the important role played by microenvironmental ECM signals for determination of stem cell differentiation fate.5,13
The growth factor content in the decellularized liver matrix of the present study showed that this decellularization protocol can preserve up to half of the HGF and bFGF content of the normal liver. HGF and bFGF are essential growth factors in angiogenesis and hepatic differentiation.29,30
VEGF content was below detection amounts in the current decellularized liver matrix.
Eventual replacement of the entire sophisticated liver architecture will require addition of liver nonparenchymal cells, especially liver sinusoidal and macrovascular endothelial cells, to vascularize the entire graft and prevent thrombosis caused by direct exposure of the collagenous basement membrane to the circulation. Moreover, strategies to solve bile drainage must be developed; however, the fact that the biliary tract is preserved by the present decellularization method maintains the possibility to reconstitute it with biliary epithelial cells.
Different techniques were investigated to seed primary hepatocytes in the decellularized liver matrix. The multistep perfusion technique produced ~90% cell engraftment efficiency. The perfusion technique showed deposition of the primary hepatocytes within the parenchymal space and occasionally in the vessels. A possible explanation for this result is the lack of the endothelial cell barrier, abundant empty parenchymal space, and the openings within the collagen-rich basement membrane structure of the vasculature due to the mechanical pressure of the perfusion flow. After engraftment, hepatocytes showed viability, albumin expression, and secretion abilities and metabolic activity in perfusion for up to 7 days. Differences in outcomes within the cell seeding techniques may be the result of different mechanical stress of each seeding technique.
Ongoing studies are directed toward complete re-endothelialization, biliary tract reconstruction, and hepatic differentiation and reconstitution using a variety of stem cells and differentiated cells. Addition of nonparenchymal liver cells and transplantation in special liver-reconditioned animal models may enhance the reconstitution and regeneration of the decellularized/recellularized liver graft in vivo. Thus, the possible in vivo production of liver grafts using decellularized/recellularized liver graft ultimately as an in vivo model for liver regeneration and development is envisioned. This technology could have a notable impact in the clinical setting as the whole-organ grafts could be used to treat patients with congenital enzymatic liver diseases, possibly allowing the repopulation of the patient liver with healthy hepatocytes. These grafts could change living-donor liver transplantation in that left-lateral grafts—currently not deemed sufficient for adult recipients—could be sufficient to support the patient when performed in tandem with the hepatic support provided by the liver grafts. This system may prove to be a superior liver cell culture system for pharmacology, drug discovery, and toxicologic studies of a compound in human liver grafts in vitro prior the exposure to the whole body, by providing a natural environment for the hepatocytes. Such utility potentially translates into reduced costs and time in drug development, and less harmful patient exposure in clinical trials. Additionally, the whole-organ liver would provide a feasible model to study liver development and ultimately as an auxiliary liver graft for transplantation.
In summary, the data presented herein support this scaffold plus cell-based regenerative medicine strategy for functional liver replacement. The resultant liver matrix scaffold has all the obvious necessary microstructure and extracellular cues for cell attachment, differentiation, functioning, and vascularization.