| Fulminant hepatic failure (FHF) is a serious clinical condition that is associated with highmortality. Hepatocyte transplantation has been considered as one of efficient ways in the treatmentof FHF.Allogeneic hepatocytes are the important cellar courses for xenotransplantation,however, Thesame problems would occur if the immunosuppressive therapies were applied to xenotransplants orfrom the use of cells that are not autologous to the patient. Microencapsulation was proposed as ameans to protect the enclosed cells from the external environment, thereby helping us to preventrejection by the host immune system. Smaller molecules, such as oxygen, nutrition and smallproteins, can rapidly equilibrate across the artificial cells.In this study, we made two types of microcapsules, the alginate-polylysine-alginate (APA) andBa-alginate-polylysine-alginate (BPA) microcapsules, and evaluated their mechanical propertiesand biocompatibilities to find an ideal one for cell transplantation. After that, we transplanted themicroencapsulated hepatocytes or co-encapsulated with sertoli cells in vivo in rat to treat FHF totest the possible use of the microcapsules we made in disease treatment. Through this work wehope to improve our microcapsule preparation protocol as well as the therapeutic effect ofmicroencapsulated cell transplantation, thus providing the theoretical and experimental basis for thefurther study and clinical applications of islets microencapsulated technology. The detailed contentsare as follows:1. Preparation and optimization of microcapsulesBy adjusting various factors of static electricity drop method, we optimized the microcapsulepreparation condition. At the request of different transplantation protocols, we successfully mademicrocapsules with controlled size that are uniform in diameter and smooth in walls. Forhepatocytes transplantation, microcapsules was made in a condition of 6kV/1.5cm of electrostaticfield, 15ml/h of pump rate, 0.25mm of inner diameter, 2％of alginate density, 0.25％of PLL densityand 6min of PLL reaction time, and the resulted diameter is 300-400μm.2. Evaluation of mechanical properties and biocompatibility of microcapsules1) The mechanical strength of different type microcapsules was tested with mechanical shaking experiments, and the osmotic pressure was measured by long-time culture in simulatedbody fluids. The results showed that BPA microcapsules have higher mechanical strength andlong-time stability than APA microcapsules.2) Transplantation strength and biocompatibility of the capsules were measured bytransplanting the capsules in the muscle and peritoneal cavity. After extracted from muscle, theAPA capsules were out of shape, swelled and had obvious fibrosis, while BPA capsules were stillspherical and only slight fibrosis was observed. These results showed that BPA capsules werestronger than APA capsules in transplantation strength. When retrieved from rat peritoneal cavities8 weeks post-graft, 76.4±8.4％of APA capsules were intact and 3.6±1.3％were wraped byperitoneal cells; 79.4±7.9％of BPA capsules were intact and 3.5±1.7％were with adherence ofperitoneal cells. These results suggested that both of the two kinds of capsules were similar inbiocompatibility.3) For the detection of microcapsules as immunoisolating barrier, the permeability ofmicrocapsule membrane was measured by Dextran with different molecular weight (MW). Theresults showed that the membrane of the two kinds of microcapsules has a similar MW cut-offrange: 4kD Dextran could pass through the memberane freely, 70Kd Dextran could pass in part,while the 150kD Dextran could not pass it. Even the smallest IgG proteins are 150 kD in molecularweight, therefore, the microcapsules we made could serve as the immunoisolating barrier andsustain the survival of encapsulated cells.3. Seperatation and culture of rats primary hepatocytes and preparation formicroencapsulated hepatocytes1) Hepatocytes were prepared by the modified methods which were in situ 2-stepcollagenase perfusion technique as described by Seglen. If the viability of hepatocytes was under90％after isolation as quantified by a 0.4％Trypan Blue exclusion test,they should be purified byPercoll solutions.2) We make the microencapsulated hepatocytes, then take the culture medium of them andthe dissociative hepatocytes to test the content of albumin. The results shows that there is not muchdifferences in the quality of albumin in the two diffient in vitro mediums. And we observate themicroencapsules with microscopes in the cultivated process, found that both of themicroencapsules and the hepatocytes have a shape of integrity. So we can conclude that the BPAmicroencapsules do not affect the functions of hepatocytes.4. Using the microencapsulated hepatocytes to cure the model of liver failureThe liver failure will induce the death of hepatocytes abundantly, when the hepatocytes dead,all kinds of enzymes will release into the blood and then will increase the guideline of the enzyme.We transplant the empty microcapsules, microencapsulated hepatocytes, dissociative hepatocytesand co-encapsulated hepatocytes and sertoli cells to the different models of liver failure rats. Wetake the blood from the vein of the eyes, and test the quality of ALT, AST and ALB in 1d,3d,7d,2w,3w,4w,5w,6w. The result shows that the co-encapsulated group is higher than single encapsulated group, and single encapsulated group is higher than dissociative hepatocytes group.So we testfiy that the function of immunity of sertoli cells protect the role of hepatocytes. And theBPA capsules also played a role as immunoisolating barrier, to improve the function of hepatocytesin the capsules.These results show that the BPA microcapsule not only has the same permeability andbiocompatibility as APA microcapsule,but also has the highly transplantation strength. Themicroencapsulated hepatocytes, co-enapsulated hepatocytes and sertoli cells transplantationshowed a significant therapeutic effect on liver failure rats.
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