Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, Shulman C, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med 2010;16:814-820. (Reprinted with permission.)
Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease.
Whole organ transplantation remains the definitive treatment option for many forms and causes of end-stage organ failure, such as chronic kidney diseases, heart failure, and liver cirrhosis. However, patients who undergo transplantation face several problems, such as surgery-related mortality and severe infections related to lifelong immunosuppression.1 Furthermore, the number of needed organs is still outpaced by the number of donated cadaver organs, and this has resulted in a high mortality rate for prospective transplant recipients and long waiting times.2 For this reason, new cell sources or alternative methods are needed.3 Many investigators have evaluated the possibility and efficiency of parenchymal cell transplantation in the form of single-cell transplantation,4 cells placed on or within matrices, and cells seeded in bioartificial organ scaffolds.5 In this way, the transplantation of isolated hepatocytes can lead, at least in rodent models, to complete organ repopulation.6 However, no attempts using matrices or other artificial scaffolds have been successful so far because their cell-harboring capacity is simply too small to substitute for liver functions over the long term.
In humans, hepatocyte transplantation has already become a clinical reality, although its status is still very experimental, and the first clinical trials have just started. In single cases, it has been shown that metabolic liver diseases (e.g., Crigler-Najjar syndrome) can be treated by hepatocyte transplantation7; long-term treatment benefits, however, still have to be demonstrated.8 Hepatocyte transplantation can also help us to use cells from donor livers more effectively because cells from one organ can be distributed to several recipients.9
However, the success of the engraftment of transplanted hepatocytes is quite variable. It sometimes leads to portal hypertension or cell embolization in the lungs.3 Investigators have thus searched for ectopic sites for hepatocyte engraftment. Therefore, hepatocytes have been transplanted into the pulmonary capillary bed, subcutaneous tissues, the renal subcapsular space, the peritoneal cavity, and the spleen10 as single cells or as two- or three-dimensional bioengineered liver systems. However, inconsistent long-term viability, small numbers of transplanted cells, and insufficient support of the cells with oxygen and nutritive substances have limited the success,3, 5, 11 even though the functionality of an implanted extrahepatic liver-assist device has recently been demonstrated in a mouse model of acute liver failure.12 In scaffold-based devices, the requirements of high blood flow rates and bile secretion into the intestines are unsolved problems over the long term.8
So far, none of the used scaffolds have resembled the three-dimensional architecture of the liver lobe; thus, the lack of the exact anatomical structure of the liver most likely prevented the long-term success of all previous attempts at creating an artificial organ. Inspiration in this respect came from another angle: in 2008, Ott et al.13 created a bioartificial heart using a decellularized heart matrix according to a detergent-based perfusion strategy. This heart scaffold could be reseeded effectively with cardiac and endothelial cells and showed physiological myocardial functions.
On the basis of these techniques, Uygun et al.14 transferred their model to the liver (see Fig. 1). They used decellularized liver matrices of rats to create a natural scaffold for seeding hepatocytes. Within a stable microvascular network and a honeycomb structure along the sinusoids (observed by electron microscopy), donor hepatocytes were successfully seeded into these liver matrices. Over time, the seeded cells migrated from the vascular side into the empty spaces of the sinusoids that were previously used by the old hepatocytes. The authors suggested that the absence of the epithelium as a natural barrier benefited this migration. With this method, they were able to recellularize a rat liver matrix with up to 200 million hepatocytes. Seeded cells were shown to have long-term viability in vitro and to exert physiological functions such as albumin and urea production. The expression levels of cytochrome P450s were also similar to those of cells cultured in established collagen sandwich cultures. Ultimately, the authors were able to transplant such recellularized liver matrices into living rats. After 8 hours in vivo, these neolivers still showed metabolic activity without greater ischemic damage to the cells.
Their article makes no reference to the reason that they left the neolivers for only 8 hours in the rats and used an ex vivo whole blood perfusion system for longer experiments.14 As in previous studies, the absence of nonparenchymal cells such as endothelial, immune, and biliary epithelial cells in the grafts is still an unsolved problem. However, in a preliminary test, Uygun's group seeded microvascular endothelial cells into the repopulated graft, and these were integrated into the remaining vascular scaffolds; this raises the hope that neovascularization of the matrix might be possible.
Thus, this is a seminal technique for creating a point of origin from which it might be possible to build artificial liver grafts. Different donor sources are possible and need to be investigated. Here Uygun et al.14 took allogenic hepatocytes from homozygote donor rats. Other cell reservoirs could be autologous tissues, xenografts, or allogenic cells stored by cryopreservation.5, 15 In the last case, the system might be a useful bridging treatment for transplantation or an alternative strategy for treating severe liver failure until the endogenous liver recovers. Until this becomes real, the system could have great value for pharmacological and virological studies if humanization of the matrix is possible.
However, several issues have to be resolved. Because the liver grafts present a lot of prothrombotic, subcellular molecules, the rats still have to be fully anticoagulated after transplantation. Sufficient re-epithelialization of these prothrombotic structures could potentially solve this problem. It has to be further determined whether the implanted hepatocytes can be supplied with enough nutrients and oxygen within their neomatrix over the long term. Moreover, an efficient bile drainage system must be reestablished. Additionally, other nonparenchymal cell fractions, such as stellate and Kupffer cells, have to reside in the neolivers and show proper functioning.
In conclusion, Uygun et al.14 have presented a technique that opens a new field in liver research and transplantation. It is now possible to generate re-implantable artificial liver grafts in rats. Although this method is still in its beginnings, it opens a big window of opportunity for further investigations.