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Contemporary Liver Transplantation provides a comprehensive review of the most crucial The Successful Liver Transplant Program Print + eBook ,79 €. ISBN: (ebook) $ Add to cart The field of liver transplantation has undergone tremendous change and continues to evolve at a fast pace. MEDLINE Abstracts: Adult-to Adult Live Donor Liver Transplantation. Read "Liver Transplantation Clinical Assessment and Management" by available from Rakuten Kobo. Sign up today and get $5 off your first download. **Liver.
The infrahepatic vena cava IVC was isolated from the inferior margin of the liver to the level of the renal veins. The liver was then retracted ventrally and superiorly, and the posterior diaphragmatic attachments of the liver were divided. The hepatoduodenal ligament was opened, the common bile duct was isolated, and Safeed Tube 2. Next after the splenic artery and the left gastric artery are ligated and divided, the celiac artery and the portal vein were isolated. Heparinized 0.
After the saline infusion, the liver was removed and weighed. The decellularized livers were washed with PBS for 1 h. The liver bioscaffold was sterilized in 0.
Cell yield was 1. Cells 0. For 3D hepatocyte culture control, 0. After cell attachment, monolayers were overlaid with ice-cold medium containing 0. Medium was changed every day. Polymer mixture 20—40 ml , depending on the liver size, was injected via inserted tubes of the infrahepatic vena cava, portal vein, hepatic artery, or biliary tree.
The decellularized liver matrix was cut into small pieces and placed in centrifuge tubes. Five samples were taken from each lobe; right lateral, right median, left median, and left lateral lobe. Specifically, each lobe was dissected into two pieces vertically along the main portal vein, and the samples were taken one from the edge, one from the surface, two from the center of the lobe close to the portal vein, and one from the proximal edge close to the porta hepatis.
DNA content in the liver matrix was determined according to Gilbert et al.
The values were normalized by cell number. Decellularized liver samples were fixed in 2. The fixed samples were washed three times with fresh buffer.
The samples were visualized using S field emission scanning electron microscope Hitachi, Tokyo, Japan. Normal fresh porcine liver was used as control. Quantification was performed in at least 10 random places on each sample. Prior to the perfusion culture experiments, we performed ultraviolet irradiation to the liver scaffold for 1 h for decontamination, and the decellularized liver was transferred to a customized organ culture chamber, which was specifically constructed for a large-scale organ perfusion; the perfusion system was designed based on previously developed system for rat liver 29 that consisted of a peristaltic pump, bubble trap, and oxygenator.
The system was placed in an incubator for temperature control, and the oxygenator was connected to atmospheric gas mixture. The medium was changed daily. To calculate the engraftment efficiency, the perfusate was collected, and the number and viability of cells not retained in the liver was determined with a hemacytometer and trypan blue exclusion. The total number of cells retained in the decellularized liver represented the difference between the initial number of cells seeded and the number of cells present in the perfusate after seeding.
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To determine if the decellularization protocol was feasible in large livers, we used native whole porcine livers, which are similar to human in size and anatomy. The decellularization protocol consisted first of a freezing—thawing technique for at least 12 h to induce cellular lysis. The whole organ decellularization was achieved then by portal perfusion with SDS, which is an anionic detergent that simultaneously can lyse cells and solubilize cytoplasmic components.
The protocol was based on the rat liver decellularization protocol that we previously developed 29 and is founded on the work published by Ott et al. Figure 1a—e shows macroscopic images of liver prior to decellularization and after various steps in the decellularization process. This protocol could create an acellular scaffold of porcine liver, which retained the gross shape of the whole organ.
Whole-organ porcine liver homogeneous decellularization. Representative images of porcine livers during decellularization process at a 0 h, b 18 h, c 48 h, d 72 h, and e 96 h. Histological comparison of normal liver and decellularized liver matrix: Scale bars: Immunological reaction of the remaining materials of the decellularized liver matrix has to be avoided if further clinical application is intended in order to elude any inflammatory reactions As porcine livers have a much larger tissue density and area, we analyzed the DNA content of the different areas and lobes in order to measure the homogeneity of the decellularization process.
Samples involved the right lateral, right median, left median, and left lateral lobe of the decellularized whole liver Fig. DNA content was decreased from Extracted DNA was quantified by agarose gel electrophoresis, which showed smearing of the fragmented DNA bands from decellularized liver samples Fig.
It is essential to maintain a three-dimensional structure of the engineering bioscaffold to generate further transplantable liver grafts, especially the vascular network and bile duct tree are important features for subsequent recellularization and surgical implantation.
The intricate vascular network portal veins, hepatic arteries, and hepatic veins remains intact as assessed by the corrosion cast of the liver bioscaffold similar to normal porcine liver Fig. Demonstrating clearly that those vessels retained their luminal diameter and shape.
In addition, bile duct structures were mostly intact; however, we observed minimal disruption of the smaller branches Fig. We performed digital radiographic evaluation of the biliary tree demonstrating that the larger bile ducts were well enhanced by the infusion of the contrast agent as if they had a natural structure; however, the localized retentions of the contrast agent were observed in the peripheral ends of the biliary tree of decellularized livers, while no retentions were seen in those of normal porcine liver Fig.
To evaluate the integrity of the microvasculature and surface capsule integrity, heparinized blood perfusion via portal vein was perfused and demonstrated that a vascular network retained intact inside of the livers and the flow moved through structures consistent with the hepatic veins; moreover, no leakage was determined from the liver surface, in opposition to what has been observed in rat livers Fig.
Decellularized liver matrix preserves entire vascular system and bile ducts. Representative photographs of decellularized left lobes of porcine liver, with the vascular tree visible.
Comparison of normal liver and decellurarized whole-liver bioscaffold of corrosion cast of a portal vein lighter staining and hepatic artery darker staining and b hepatic vein and bile duct white as well as by c radiographic imaging of the bile ducts.
Histological analysis of the decellularized liver, especially Azan staining, revealed that collagen fibers were well retained in the decellularized scaffold showing the small lobular components in detail Fig.
In addition, immunolabeling characterization of the liver ECM showed the preservation of collagen type IV, fibronectin, and laminin, indicating a similar structural composition of the matrix to that of intact porcine liver Fig. Ultrastructural characterization of the decellularized liver matrix was performed using scanning electron microscopy SEM images comparing to porcine normal liver Fig.
To generate transplantable liver scaffold, it is critical to keep the integrity of the surface i. We evaluated impairments of the surface on the decellularized structure meticulously and found that most of the liver surface area was intact 1. Sections were counterstained with DAPI. To determine if the decellularized native liver derived bioscaffold could provide a suitable environment for cells by providing not only a three-dimensional structure but also maintaining bioactive molecules, we evaluated the expression of the growth factors such as HGF, bFGF, VEGF, and IGF-1 in the scaffold.
Those factors are essential for liver regeneration and angiogenesis. While HGF and bFGF was highly expressed on the cells in the parenchyma and on the cells in the vessel walls, respectively, in normal liver, there was a lower expression in the scaffold even after the decellularization. Interestingly, VEGF, which is mainly released from endothelial cells as one of the critical growth factor for angiogenesis, was also observed in the acellular matrix 4.
In addition, IGF-1, which is released from hepatocyte, was also observed in acellular scaffold. The expression in acellular scaffold 1. Decellularized whole-liver scaffold retained growth factors expression.
(ebook) Liver Transplantation: State Of The Art
Immune-histochemical evaluation of growth factors. Immunostain without primary antibodies with secondary antibodies served as controls in each experiment.
To determine if porcine primary hepatocytes could efficiently be delivered in this large-scale liver bioscaffold, an organ chamber was manufactured that contains a sterile main reservoir in which the decellularized liver matrix is mounted Fig. Twenty four after the infusion, more than half of the attached cells in the decellularized liver scaffold attached cells: Porcine livers Fig.
Albumin staining after 4 and 7 days of culture-perfusion showed that hepatocytes engrafted around the larger vessels, repopulating the surrounding parenchymal area. The amount of immunostaining albumin of engrafted hepatocytes after 4 days of the perfusion culture was similar to that in normal livers; however, the expression decreased considerably after 7 days in the perfusion culture Fig.
The protein synthesis of infused cells was analyzed by the detection of albumin and urea concentration in the perfusion medium, compared to those in cultured porcine hepatocytes on collagen single layer. The albumin secretion Fig. Although the protein synthesis was relatively higher in the perfusion-cultured hepatocytes, there was no significant difference in two groups for up to 7 days.
However, when hepatocytes were cultured in a double-gel configuration Fig. Double-gel cultured porcine hepatocytes demonstrated a superior albumin secretion and urea synthesis ability when compared with perfusion-cultured porcine hepatocytes or porcine hepatocytes cultured on collagen.
Recellularization of a large-scale liver scaffold using porcine primary hepatocytes. The present study describes a minimally disruptive method for whole-organ decellularization protocol in a large animal model that produces a three-dimensional liver matrix suitable for supporting functional hepatocytes, a broad characterization of the decellularized liver matrix shows maintenance of the hepatic matrix components and preservation of the vascular network, bile duct tree, and representative growth factors such as HGF, bFGF, VEGF, and IGF The results of the present study not only confirm the concept of whole organ decellularization, as previously shown 5 , 19 — 21 , 24 , 27 — 29 , but also provides methodology of organ engineering in a large size-scale adapted from previous rodent studies that will be important for clinical translation of this regenerative medicine approach for liver replacement.
To obtain a completely acellular porcine liver three-dimensional scaffold, we optimized a protocol that builds on the technique developed for heart perfusion decellularization by Ott et al. This decellularization protocol showed i preservation of the three-dimensional architecture, the native structural and basement membrane matrix, as well as the functional vascular network of the original organ including the biliary network; ii homogeneous and complete decellularization quantified by DNA content criterion in all the different segments of the liver; iii growth factor content can be preserved within the matrix scaffold at a level comparable to the native liver; and iv multistep cell seeding method showed high-efficiency engraftment of functional hepatocytes using a customized organ culture system.
Whole-organ decellularization has been recently reported for different organs including liver 5 , 24 , 28 , 29 ; however, these whole-organ decellularization techniques have been scaled up to a clinically relevant size only for heart 30 ; moreover, most in vivo implantation in rodent studies has been limited to a few hours due to problems such as hemorrhage, thrombosis, and surgical technical difficulties.
The method for whole-liver decellularization reported herein using porcine livers tackles and facilitates some of the issues e. Post decellularization process, the organs were translucent while preserving the three-dimensional architecture; moreover, immunostaining suggested that the matrix of both the large vessels laminin and the sinusoids collagen type IV, fibronectin remained intact.
Transplantation of the Liver
Furthermore, portal vein blood perfusion, corrosion casting, digital radiographic evaluation of the biliary tree, and SEM analysis demonstrate the presence and function of the microvascular network of the liver post decellularization. Moreover, growth factors and chemoattractants are immobilized by ECM components through disulfide bonds 1. Thus, the precise ECM arrangement present in the adult liver could potentially facilitate and guide the developmental pathway for full hepatic maturation of stem cells.
Thus, decellularized organs could potentially become a tool for stem cell differentiation and maturation to eventually engineer autologous liver grafts for transplantation Liver cell sources to engineer liver grafts can primarily be obtained from livers rejected for orthotopic liver transplantation, unused segments of donor livers, and non-heart-beating donors.
But, these sources do not begin to approach the potential numbers needed to treat all patients that might potentially benefit from auxiliary liver transplantation. Ideally, stem cells derived from the subject to be treated would be the perfect cell source to engineer personalized liver grafts.
Induced pluripotent stem cells iPS cells have been created by transducing mature cells with transcription factors that transform them into cells that have characteristics and differentiation potential nearly identical to human embryonic stem cells ES cells.
While there is much enthusiasm for the potential use of stem cell-derived hepatocytes, generation of a sufficient mass of functional hepatocytes for treatment of liver failure from autologous cells derived from iPS cells would require a period of weeks for expansion, differentiation, selection, and testing to exclude contamination by tumorogenic precursors, far too long to address the problem of acute hepatic failure.
These factors are essential in angiogenesis and hepatic regeneration and differentiation 8 , 12 , In the present study, the decellularized native liver matrix was able to retain such growth factors after the whole decellularizing process, which may provide sufficient environmental cues to cells for survival and function.
However, continuous production of angiogeneic factors e. Eventually rebuilding the entire sophisticated liver architecture will require addition of liver nonparenchymal cells, especially liver sinusoidal and microvascular endothelial cells to vascularize the entire graft and prevent thrombosis caused by direct exposure of the collagenous basement membrane to the circulation.
In addition, biliary epithelial cells would be necessary in order to solve bile drainage. The perfusion technique showed deposition of the primary hepatocytes within the parenchymal space and occasionally in the vessels. However, future studies will be performed in order to improve stability of hepatocicyte survival and function to achieve comparable function of double-gel culture hepatocytes e.
Addition of nonparenchymal liver cells to reconstitute the sophisticated anatomy of the hepatic sinusoids are necessary, but the ultimate test is the transplantation in relevant animal models of liver failure;, thus, reliable models of liver failure in large animals will have to be developed.
In summary, the data presented herein provide a foundation for efficient development of clinically relevant liver scaffold and a cell-based regenerative medicine approach to build liver grafts for transplantation.
The authors thank Mr. Toshio Otake, Mr.
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If you have not received any information after contact with Australia Post, please contact us to confirm that the details for delivery logged with us are correct. We will then contact you with the appropriate action.Disease Recurrence After Liver Transplantation.
Outcome Predictors in Transplantation Helen E. English Download options: Philip Lanzkowsky.
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