Decellularization is the most promising near term approach to generating patient-matched organs for transplantation. It is a fairly simple concept at root: researchers remove all of the cells from an organ, leaving the scaffold of the extracellular matrix with all of its intricate details and chemical cues. The challenge lies in building a reliable methodology that can be scaled up for widespread use. Much of the work on decellularization to date has focused on hearts and lungs, but in the paper noted here, researchers outline a method for reliably decellularizing whole livers.
Decellularization does of course require a donor organ as a starting point, unfortunately, but that can include a significant fraction of the potential donor organs that would normally be rejected by the medical community for one reason or another, as well as organs from other species, such as pigs. Given suitably genetically engineered pigs, a decellularized pig organ repopulated with human cells should contain no proteins that will provoke significantly harmful responses following transplantation. This and other options should roll out into availability in the years ahead, ahead of the range of more ambitious tissue engineering projects that aim to grow entire organs from a patient cell sample.
Decellularization is ahead of other methodologies for the creation for patient-matched organs because the research community has yet to produce a good method of generating the intricate networks of tiny blood vessels that are needed to support tissue much larger than a millimeter or two in depth - the distance that nutrients can perfuse in the absence of capillaries. Yet over the past few years many research groups have demonstrated the production of organoids, tiny sections of complex, functional organ tissue, for a variety of organs. Thus the actual production of organs from patient cells will be a going concern just as soon as the blood vessel question is figured out. Unfortunately, this has been the state of the field for years now, with many promising leads but no definitive end in sight. Meaningful progress in bringing decellularization to the medical community is to be welcomed in the meanwhile.
The only therapy for liver cirrhosis is liver transplantation, but the shortage of organ donors imposes a severe limit to the number of patients who benefit from this therapy. With increasing shortage of donor organs and decrease of their quality, the development of novel procedures and alternatives for organ transplantation becomes essential. Thus, organ engineering, which involves the repopulation of acellular matrices, was explored with the use of polymeric scaffolds or three-dimensional (3D) printing of liver tissue to make scaffolds that can be seeded with hepatocytes or other cell types.
Although these are powerful tools worth exploring, it remains difficult to design and create artificial, yet functional liver tissue with functional vascular and biliary trees for clinical use. Alternatively, removal of cells from an existing organ, leaving a complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM), may provide a natural habitat for reseeding with an appropriate population of cells, and connected to the blood stream and biliary system.
Ideally, ECM is cell free, but remains the interlocking mesh of fibrous proteins (collagen, elastin, fibronectin, and laminin) and glycosaminoglycans (GAGs). Evidence from rodent models shows the feasibility of decellularization of whole liver organs that provides an excellent scaffold for reseeding liver (stem) cells for graft engineering. Also, porcine and sheep liver have been successfully decellularized to obtain ECM for transplantation. However, so far, there is very limited experience with decellularization of whole livers from humans.
Recently, researchers demonstrated efficient decellularization of a whole liver and partial livers to generate small cubes of human liver scaffold. Different decellularization methods have been described among which are physical force (freeze/thaw, sonication, and mechanical agitation), enzymatic agents (trypsin, endonucleases, and exonucleases), and/or chemical agents (ionic, nonionic, and zwitterionic detergents). Usually, combinations of these methods are used. In larger organs, such as human or porcine liver, perfusion through the intrinsic vascular beds is the favorable route to be able to reach all cells. So far, most experimental decellularization protocols include the use of sodium dodecyl sulfate (SDS) to generate full freedom of cells and translucency, but this also progressively destroys the ECM and hampers clinical translation.
In this study, we report successful decellularization of human livers to obtain transplantable whole organ scaffolds. We show proof of concept that these scaffolds can serve as feasible resources for future tissue-engineering purposes. Using a controlled perfusion system, a complete 3D acellular human liver scaffold was generated on a clinically relevant scale and free of allo-antigens. We present the feasibility of systematically upscaling the decellularization process to discarded human livers. Eleven human livers were efficiently decellularized by nonionic detergents by machine perfusion. A careful choice of the decellularization methodology is of great importance as methods described for decellularization may be well suitable for other organs than the liver, but may damage the composition of the matrix proteins.
Repopulation of a complex organ such as the liver poses numerous challenges. Using the extracellular matrix of the native liver obviously helps to create the most optimal niche for cells to repopulate, but the types of cells to be infused to create fully functional liver tissue remains to be elucidated. In addition to the liver-specific matrix proteins, the still present vascular and biliary system may also provide entry routes for the different cell types needed. Obviously, efficient recellularization is a complex process in which hepatocytes or other parenchymal cells need to pass the remnant basement membrane of the decellularized blood vessels or bile ducts to enter the parenchyma after vascular or biliary administration, respectively. In addition, cell numbers that are required for efficient recellularization are highly dependent on cell type and volume of the scaffold.
Reendothelialization is a pivotal step to prevent thrombosis as a result of the massive collagen contact surface that blood will encounter upon reperfusion, and which cannot be prevented by coating with heparin. We demonstrated, like others did in animal models, that matrix sections can be reseeded with endothelial cells and these cells end up at the location of the decellularized blood vessels and pave the basal membrane. In our studies, HUVEC were used as a source of endothelial cells, as in most studies in rodents and pigs, but other sources such as endothelial progenitor cells are also used and show similar results. The next hurdle to be taken toward clinical application is to choose a cell source for liver parenchyma repopulation. An adult liver contains ∼150-350 billion cells of which the largest part (70%-85%) is made up by hepatocytes. However, adult primary hepatocytes of high quality are scarce and therefore limit tissue-engineering applications. Ideally, autologous cells, isolated from the patients themselves, are used as these cells will have a low risk to trigger an immune response. Alternatively, (autologous) pluripotent stem cells that self-renew and are able to differentiate into all cell types needed could be seeded.
In summary, human cadaveric livers can be successfully decellularized using machine perfusion and nonionic detergents, and can be repopulated with endothelial cells. The next steps toward clinical application involve finding a cell source or combinations of cell types to reseed the matrix, including the vascular and biliary system, to gain functional liver tissue.