These results present the first step toward development of recellularized liver matrix as an auxiliary graft for transplantation. Previous attempts, including those of our group, have failed in creating complex tissue-engineered constructs for liver transplantation16,18
. Here we successfully demonstrate the preservation of the liver’s three-dimensional architecture, functional vasculature and native matrix composition upon decellularization. Further, we present a technique for efficient in vitro
recellularization of the graft, which maintains cell viability and function. Finally, we demonstrate the feasibility of transplanting these recellularized liver grafts in vivo
with minimal ischemic damage.
Oxygen and nutrient transport limitations have thus far restricted the development of tissue-engineered liver constructs to the diffusion limit, about 200 μm. The high rates of oxygen consumption33–36
by primary hepatocytes result in ischemic damage in cells found further away from the blood within minutes of transplantation. Owing to high oxygen demands, the liver is the only organ that receives both arterial and venous blood supply, whereas hepatocytes are located less than 2 μm distance from the nearest blood vessel17
. It is a crucial configuration that should ideally be replicated in vitro
This report builds on the previously reported technique developed for heart perfusion decellularization27
, demonstrating its applicability for rat liver decellularization and further extending its innovative contributions by introducing a perfusion-seeding method and an extracorporeal perfusion-culture system. Additionally, we further optimize the decellularization for liver.
Our work shows that portal vein perfusion decellularization creates a translucent liver matrix while preserving the three-dimensional architecture, the native structural and basement membrane matrix, and the functional vascular network of the original organ. Portal vein dye perfusion, corrosion casting, SEM and immunostaining show the presence and function of the microvascular network of the liver post decellularization. Quantification of extracellular matrix proteins, SEM analysis and immunostaining suggests that the matrix of both the large vessels (collagen type I, laminin-β1) and the sinusoids (collagen type IV, fibronectin) remained relatively intact. We note that the main structural protein of the liver, collagen type I, was retained in its entirety during the decellularization process. However, we also note the marked loss of half of the glycosaminoglycan content of the liver during decellularization. This loss is not surprising, as many glycosaminoglycans are associated with cellular membranes that are solubilized in the decellularization process.
We have found that the DLM can be seeded with primary rat hepatocytes via a multistep perfusion technique, with efficiency greater than 90%. As expected, this grafting efficiency is much higher than in vivo
levels of engraftment after hepatocyte transplantation, possibly as a result of the lack of endothelial barrier and the abundant empty space. We seeded 50 million primary rat hepatocytes, roughly 5% of the cells in native rat liver, as this amount was previously shown to be sufficient to restore liver function in animal models6
. We have also shown that this number can be increased up to 200 million to account for 20% of the native rat liver hepatic mass, corresponding to repopulation of approximately 50% of the original hepatic mass within the lobe used for transplantation. After engraftment, hepatocyte viability and metabolic function was maintained in perfusion culture, as assessed by TUNEL staining, LDH release, synthetic activity and gene expression. Engrafted cells showed albumin production and gene expression equivalent to cells from the same isolation cultured in a collagen-sandwich configuration, a culture configuration previously shown to maintain high liver-specific function for up to a month in vitro37
. Overall, albumin production and gene expression were roughly 20% and 30% of in vivo
We have shown that rat livers can be depleted of their cellular components and used to engineer sophisticated hepatic structures that may recover many functions of normal liver. Despite its advantages, further improvements of the decellularization and recellularization of liver matrix are necessary. Engineering of an entire liver will require addition of nonparenchymal cells such as liver sinusoidal endothelial cells, stellate cells, biliary epithelial cells and Kupffer cells. Particularly, we observed the presence of hepatocytes outside the microvasculature system, mainly distributed in the parenchymal spaces, possibly owing to loss of sinusoidal integrity in the absence of the liver sinusoidal endothelium and other nonparenchymal cells. We speculate that the nonparenchymal cells of the liver will be essential for reacquisition of vascular integrity. Our ongoing studies are directed toward optimizing seeding strategies, re-endothelialization and biliary tract reconstruction. However, our preliminary results on re-endothelialization of the graft indicate the feasibility of this approach. Addition of nonparenchymal cells and modification of the perfusate may further enhance the reconstitution of a functional liver graft and even lead to hepatic repopulation by imitating the regenerative behavior of the liver. Thus, we envision the possible use of DLM ultimately as a model for the study of both liver development and liver regeneration.
In summary, this report provides a foundation for efficient development of auxiliary liver grafts for transplantation and practical, unique techniques to prepare recellularized liver graft. Further studies are required to determine whether the techniques described here can be scaled up for use in humans.