Construction Of Engineered Liver Tissue Based On The Coculture Of SC And Hepatocyte

Since liver is one of the vital organs of human body, liver diseases have severe effect on patients'health and quality of life. Therefore treatment of liver diseases has drawn the attention of researchers both domestic and abroad. Currently, bioartificial liver and liver transplantation is the most effective approach in the treatment of liver failure, end-stage liver disease and metabolic liver diseases. Bioartificial liver could not perform an overall liver function .Constrained by the shortage of organ supply and the immunological rejection after transplantation, liver transplantation could not be applied widely. Therefore, it is urgent to find new techniques and tools for the treatment of end-stage liver disease .The development of liver tissue engineering brought new hope for the treatment of liver diseases.In view of the complexity of the liver structure and function ,it is a great challenge to construct engineered liver invitroly . In recent years, great progresses have been achieved in liver tissue engineering research, such as: the application of three-dimentional cell assembly technology to construct engineered liver tissue, which present physiological activity in the gelatin /chitosan three-dimensional structure after cultivation for 6 days. Another scholar has designed a uniform layer of engineered liver patch, cultivated on temperature-sensitive surface. which can survive for 200 days after subcutaneous transplantation. However,the problem of immunal rejection aroused during transplantation still remained for further investigation.The mechanism for immunal rejection after organ transplantation is identified,which is the rejection of mediated by T cell and antibody.The T cell attacked the target cell mainly through two mechanisms:1.release of Fas to induce apoptosis of target cell expressing Fas-Lon celluar membrane;2.to transmit killing factors through perforin.Clinically, we applied eneral immune depressant to control the immunal rejection.However,there are disadvantegous :1.great sys-toxicity;2.dosage control;3.great expense.People are exploring new approaches to reduce the immunal rejection after organ transplantation in order to avoid the application of sys-immunal depressant.It is known to all that the didymus tissue was the native immune-privileged site . Survival rate of didymus allograft was high.Studies showed that when transplanted subcutaneously,the didymus could generate immune tolerance.Former studies focused on the didymus as whole.Recently,the didymus cells were isolated for study.The SC expressed Fas-L stably ,resulting in immune tolerance.SC could kill lymphocyte through cell to cell contaction by Fas/Fas-L; 2.kill lymphocytes and neutrophilic cell by releasing Fas-L;3.attracting inflammatory cell infiltrate and help the graft to avoid attacking.Besides,it could also secret various of growth factors providing nutritions.Furthermore,the SC adhered to grow,which make it easy for collagen contraction.Therefore,the study of SC is becoming more and more popular.2003,Rahman from England demonstrated that co-transplantation of SC and HepG2 microcapsule could increase HepG2 activity,reduce immunal rejection,and showed an improvement of liver failure.The SC was also proved to have an effect on the affected neurone.Therefore,the SC could provide nutrition to cocultured cells as well as reduce immunal rejection.There are't any report about the construction of engineered liver tissue using SC and hepatocytes as seeding cells.In this study, we isolated and cultured hepatocytes and Sertoli cells (Sertoli Cell, SC) invitroly. The morphology and function of hepatocytes and SC were detected .We also constructed engineered liver tissue with rat hepatocytes and SC in three-dimensional hydrogel. On this basis, the tissue-engineered hepatocytes /SC patch was transplanted subcutaneously in liver failure rat model. The mortality of transplanted and untransplanted ones were determined .After a certain period of time, rats were sacrificed to harvest the implanted materials for histological and immunohistochemistry assessments.Through the researches above, we successfully constructed engineered liver patch which survive well after subcutaneous transplantation, with cell aggregate into a spherical structure. The results showed that tissue-engineered liver patch is of great significance in liver transplantation.In this paper, the main research contents include the following four parts:Part I:the isolation ,cultivation and identification of rat liver cellsThis experiment used the method of portal vein perfusion, after perfusion for two times ,the hepatocytes of SD rat was derived. Immunohistochemical results demonstrated a good vitality of separated cells, cells were ALB,CK18 positive. In order to assesst the ability of glycogen synthesis and albumin secretion of the isolated hepatocytes,We used PAS staining, which result showed that the isolated hepatocytes has a capacity of glycogen synthase; in the assessment of culture medium supernatant, we also demonstrated that the liver cells have the capability of secreting ALB.Partâ…¡:isolation, cultivation and identification of Sertoli cellsIn this study, we derived SC by V-collagenase digestion. Immunohistochemical results showed a good vitality of isolated cells, cells were Fas-L positive. To study the function of SC, we prepared the SC conditioned medium which was also applied in the cultivation of lymphocytes and hepatocyte.We detected the cell apoptosis of lymphocytes through flow cytometry. The results showed that, SC could induce the apoptosis of lymphocytes. The assessment of supernatant of hepatocyte cultivated in conditioned medium showed that secretion of ALB in coculture system was increased significantly compared with hepatocytes cultured without SC conditioned media.Partâ…¢, construction of engineered liver based on hepatocytes/SCIn this study, primarily isolated rat hepatocytes and Sertoli cells were mixed with 10% Matrigel and liquid typeâ… collagen as scaffold material to construct engineered liver patch and exert statistic stretching force while in cultivation. We observed cell growth in the patch, and after a certain period of time, the patch was subjected to histological, immunofluorescent staining, as well as the function assessments. The results showed that the primarily isolated rat hepatocytes present a good vitality in three-dimensional gel matrix environment, secreted ALB, and formed a spherical structure through mutual contacts ,adhesion and aggregation.Part IV study of tissue-engineered liver transplantation in vitroIn this study, the patch constructed above was grafted into the adult hepatic failure rat abdominal subcutaneous. There were 4 groups, G1:hepatocyte/SC patch transplanted to immune depressed SD rats; G2: hepatocyte/SC patch transplanted to non-immune depressed SD rats;G3:hepatocytes only patch transplanted to immune depressed SD rats;G4:hepatocytes only patch transplanted to non-immune depressed SD rats. These four groups above were harvested 3 weeks later and subjected to histology and immunohistochemical study. The results showed that, cells in G1,G2groups survive well and attached to each other. Cells in G3 grow well with normal activity .Almost no cells survived in G4 .In conclusion, primary rat hepatocytes, SC cells were mixed with the typeâ… collagen and Matrigel to construct engineered hepatic tissue which was exerted a static stretching force during cultivation. The results showed that in the three-dimensional extracellular matrix gel, hepatocytes present good vitality, the cells from a spherical structure through mutual contacts adhesion, aggregation. The engineered liver tissue can be used as model for the study of liver development as well as drug screening. More important, this study provided a strategy for the reconstruction of other vital organs.