The Recellularization Of Acellularized Liver Scaffold With Perfusion System In Vitro And The Expermental Study Of Immunogenicity

Currently, the only solution to End-stage liver disease is organ transplantation. Transplant surgery has a high degree of complexity.High risk and was expensive.Nevertheless because of the shortage of organ source and the long-term use of anti-rejection drugs after surgery,liver transplantation can not meet the clinical need, many patients who need liver transplantation were dying in the process of waiting for the liver donor. Hepatic Tissue Engineering is a step toward alleviating the need for donor organs. Hepatocyte transplantation offers an alternative way to treat patients with liver diseases and years of laboratory and clinical studies have demonstrated its efficacy. However, a limited cell supply and low engraftment efficiency have limited this approach,At the same time, Oxygen and nutrient transport limitations remain a major concern.Bioartificial liver provides the temporary replacement of liver function with new research program Some of which has entered clinical trials stage,.Bioartificial liver system has been used as an effective means for acute liver failure (ALF) treatment, also working as a temporary liver substitution. However,being limited by the negative factors of technology, bio-safety and so on,the existing artificial liver system remained to be further improved. There are common features of many tissue-engineering projects. such as cell type, structure, and scaffold material,immunology barrier.The scaffold material benefits include providing a place for attachment, increased surface area, support for a larger cell mass, and the capability of shaping specific structures., so as to control the morphology of the cell during the procession of two-dimensional or three-dimensional culture. However, comparing with the complicated liver structure.the previously used synthetic scaffolds are all obviously too simple to meet the nutritional requirements for high-density growth of liver cells,let alone the imitation of the comlex structure and the micro-environment of liver.Therefore,it is urgent to find a kind of scaffold that can provide a three-dimensional micro-environment,which utmostly imitating the vivo environment liver cells lives.Decellularization is an attractive technique for scaffold preparation in tissue engineering, as the resulting material can potentially retain the architecture of the original tissue, including the functional aspects of the native microvasculature. The potential applications of decellularized matrix in tissue engineering have been demonstrated for a number of tissues, More recently, another group reported the decellularization of an entire heart through perfusion, preserving the original architecture and original microvascular network and allowing for extensive recellularization Solid organs have an intricate vascular tree, which through a series of branching vessels, forms a pervasive capillary network that ensures that all cells in the organ get enough nutrient and oxygen source.However, effective decellularization of thicker organs and tissues has been very difficult to achieve due to inefficient penetration of the decellularization solution into the organ. More recently, Ott et al. have developed a more effective method for organ decellularization.。They have shown that by perfusing a detergent solution through the vascular network rather than relying on agitation and diffusion alone, the entire mouse heart could be decellularized and used as a scaffold for tissue engineering The technology of decellularization uses the specific physical, chemical, and enzymatic methods to removal of cells from a tissue or an organ and leaves the complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM),The goal of a decellularization protocol is to efficiently remove all cellular and nuclear material while minimizing any adverse effect on the composition, biological activity, and mechanical integrity of the remaining ECM. The most robust and effective decellularization protocols include a combination of physical, chemical, and enzymatic approaches.which method was selected depended on the different tissue and organ.Basak E Uygun modified and applied their perfusion decellulariza-tion technique to prepare whole liver grafts, and we further introduced perfusion-seeding and culture techniques for the preparation of recellularized liver matrix for transplantation. Thomas Shupe and Pedro M. Baptista have made new breaktrough in this field.There was no standard way to make the decellularized liver matrix. Different kinds of detergengs and passways had different effect on the components of the ECM were retained.In previous work, our group has successfully made decellularized scaffold by chemical methods. The goal of our study was to develop a novel scaffold that human liver cells could readily enter in order to repopulate the scaffold volume. We report the production of such a scaffold via a decellularization processthat preserves the macrovascular skeleton of the entire liver while removing the cellular components.The intact vascular tree is accessible through one central inlet, which branches into a capillary-like network.and then reunites into one central outlet. we have combined the scaffold that preserved the pipeline structure after the decellularization into the Circulation devices constructed by ourself and co-culturing with the liver tumor cell-lines in vitro which were injected by portal vein and inferior vena cava..Experiments result confirmed that the cells could adhere to the scaffolds closely and be able to keep certain hepatecytes activity and function for a long time even in the circulating state..In addition, the xenograft implantation test result showed that the acellular scaffold has been comletely decellularized and resulted to minor minimal inflammation and good histocompatibility. The test made a basis for the further use of acellular liver scaffolds to construct a tissue engineering liver that can be transplanted.We envision the possible use of DLM ultimately as a model for the study of both liver development and liver regeneration.Further studies are required to determine whether the techniques described here can be scaled up for use in humans.ChapterⅠ:Recellularized the acelluar liver scaffold with scaffold with the circulating perfusion system.Objective:To further construct whole-liver scaffold with intact three-dimensional geometry and vasculature and bile duct by step-decreased dose inonic detergent method to.recellularize the scaffold with improved circulating perfusion system and evaluate the function expression during the circulating perfusion co-culturing test.Methods:The catheter in the portal veins were attached to a pump and EDTA solution was perfused through the portal vein at the rate of approximately10ml/minute,subsequently1%Triton-X100with1%/0.5%/0.1%SDS were perfused through the liver to decellariz the organ.Finally PBS was circulated to wash out the decellularization detergent at the rate of10ml/min,cannulated inferior vena cava and perfused sequentially with the same detergentsas control,keep the decellularized liver scaffold in the PBS solution at4℃, we introduced HepG-2cells via portal vein and inferior vena cava perfusion recirculation alternately,Rough10million cells were introduced at each step,for a total of four steps,with15-min intervals between each step,After seeding, we transferred the recellularized grafts into cell culture chamber for4hours,on the other hand,put the same amount of cells as plate culture control group.The system was placed in an incubator for temperature control and the oxygenator was connected to atmospheric gas mixture.The graft was continuously perfused through the portal vein with continuous oxygenation.The medium was changed daily.To assess the metabolic activity of engrafted hepatocytes,we quantified albumin production and urea synthesis. ALT and AST expression levels were detected to judge the viability of the cells.Result:We constructed the circulation culture system with scaffolds successfully and made7times of culturing tests.Among them,culturing pollution were found in one time in the training process.The main reasons might be for the improper operation of fluid changing in the building process and training process and so on.The cumulative urea and albumin amounts in the recellularized liver system were statistically higher than static culture control group during the5-d culture period ALT and AST expression levels were higher in recellularized liver than control groups in the first two days and lower in the later three days.Conclution:We have achieved that the DLM can be seeded with HepG-2cells via a multistep perfusion technique through the circulation pefusion system which was devised by ourselves and the feasibility of experimental design was proved.we 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.Chapter Ⅱ The xenotransplantation immunogenicity expermental study of acellular liver scaffoldObjective:To construct a whole-liver scaffold with intact three-dimensional geometry and vasculature and bile duct by graded-decreased concentration of inonic detergent method. In vivo immunogenicity was assessed by implantation of rat decellularized liver matrix subcutaneously into rabbit.Methods:take the acellular SD rat liver scaffold as research object.Cannulated the portal vein and perfused sequentially withEDTA/1%TritonX100and1%0.5%/0.1%SDS,and then perfused with phosphate buffered saline to dilute SDS residue. The morphology and Architect-ture of the extracellular matrix were examined by the macroscopic, HematoxylinEosin staining,Masson’s trichrome stainingand Scanning electron micrographs. To show that the decellularization protocol was successful in retaining a functional vascular bed, we characterized its perfusion with Red dye. the scaffold tissue were cut into slices with a diameter of about lcm and lmm thick and were immersed in peracetic acid (0.1%) on the shaker at the rate of300RPM for3hours,and then was thoroughly by stirred with enough PBS solution.Tissue samples (1cm2) were implanted subcutaneously in either side of the middorsal line. the implanted tissues retrieved for analysis at two weeks and four weeks. Keep the sterile principle during the process and sterilize the would everyday,.it is necessary to inject the rabbits with Penicillin twice a day for three days. take the same batch and weight the New Zealand big white rabbit as control group which were made the wound which was the same size. Gross observation and histological evaluation such as tissue inflammation degree were evaluated after the operationand the amount of the CRP and IgE were detected everyday.Result:During the5days after the operations,there is no greatly rise in terms of CRP and the amount of IgE nearly cannot be detected,there was no significant difference between the test group and the control group. the implanted tissues retrieved for analysis at2weeks and4weeks. The explanted acellular liver matrix samples appeared white and were soft. Finally, the specimens was dehydrated by paraformaldehyde and embedded in paraffin.HE staining and Wright stain revealed that the implanted fresh human amniotic membrane had become vacuolated and that only a small number of cells were found to be present in the junction between the implant and the muscle tissue at4weeks while there is a certain amount of cells to be found at two weeks. Masson’s Trichrome revealed that there were plenty of collagen fiber in the center of the plant.the result also indicated that the implant were not degraded obviously.Conclusion:Perfusion with detergents by portal vein for liver decellularization was an efficient method to obtained a completely decellularized whole-liver scaffold which retain low immunogenicity of the extracellular matrix components. So, the acellular liver scaffolds can be seem as a candidate for liver tissue engineering scaffolds.