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The advancement toward a clinical application for porcine islets to cure diabetes in humans must include reproducible long-term successes in non-human primate (NHP) models. Many dedicated researchers around the world are continuing to work toward this goal. In this chapter, we describe procedures for islet isolation of pancreatic islets from adult and neonatal/fetal pigs. We further include procedures for the induction of diabetes in non-human primates and subsequent insulin therapy, islet transplantation, immunosuppression, and also the daily maintenance of xenotransplanted NHPs. The procedures that we outline in this chapter are ones that we have successfully utilized in pig-to-NHP islet transplantation models. However, where appropriate, alternative methods will also be identified.Various liver diseases result in liver failure, and liver transplantation as a definite treatment is limited by the shortage of organs available for transplantation. The use of isolated primary hepatocytes in cell-based therapies including hepatocyte transplantation, tissue engineering liver transplantation, and bioartificial liver support systems has gained increasing interest during the past years. Human hepatocytes are the preferred source of cells. Aside from the organ shortage, the isolation of human liver cells is usually limited by obtaining a sufficient quantity of high-quality, metabolically active cells. Furthermore, livers from which hepatocytes are typically harvested are not suitable for transplantation, with the variability in quantity and quality. Porcine hepatocytes, on the other hand, have the ability to perform complex biological functions and show modifiable behavior. Primary porcine hepatocytes are currently widely used in the investigation of drug metabolism, hepatotoxicity, protein biosynthesis, and gene expression. Primary hepatocytes do not proliferate in vitro and are sensitive to freeze-thaw damage in cryopreservation and thus need to be freshly isolated for each experiment. Consequently, the methods of porcine hepatocyte isolation are being actively sought after. Our laboratories have been involved in various applications of liver cells, and we have long-lasting experiences in liver cell isolation and their application in R&D. We here summarize the present protocol of our laboratories for primary hepatocyte isolation from pig and their liver tissue engineering for xenotransplantation.Current treatment options are not providing an adequate solution for cartilage defects. Articular cartilage lesions in particular are not able to repair spontaneously and progressively degenerate with an arthrosic pattern. Aiming to solve this pressing medical need, xenotransplantation of porcine chondrocytes could be developed as a new therapeutic approach. Xenotransplantation is gaining much attention, thanks to the advances in animal genetic engineering and progress in the characterization of the rejection mechanisms that prevent long-term graft survival. In this regard, our team has identified various targets for intervention that should be tested in a meaningful animal model to prove their relevance in rejection of xenogeneic cartilage. To this end, we have recently established a discordant xenotransplantation model by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats. This chapter describes this new model, which can be used to assess the immunoregulatory effect of a variety of strategies designed to inhibit rejection of xenogeneic PAC both at the humoral and cellular levels.Corneal transplantation for the treatment of corneal blindness is challenging in many countries due to the shortage of graft procurement. Xenocorneal transplantation is an interesting alternative to explore despite immunologic rejection, which mainly involves endothelial cells. As anterior lamellar keratoplasty, when indicated, shows less immunologic reaction, we developed and describe below a pig-to-non-human-primate model of anterior lamellar corneal xenograft. This model can be used to assess the efficacy of corneas from genetically modified pigs.Millions of patients with valvular heart disease have benefitted from heart valve replacement since the procedure was first introduced in the 1960s; however, there are still many patients who get early structural valve deterioration (SVD) of their bioprosthetic heart valves (BHV). BHV are porcine, bovine, or equine tissues that have been glutaraldehyde fixed to preserve the tissue and presumably make the tissue immunologically inert. These glutaraldehyde-fixed BHV with anti-calcification treatments last long periods of time in older adults but develop early SVD in younger patients. HexamethoniumDibromide The consensus at present is that the early SVD in younger patients is due to more “wear and tear” of the valves and higher calcium turnover in younger patients. However, as younger patients likely have a more robust immune system than older adults, there is a new hypothesis that BHV xenografts may undergo xenograft rejection, and this may contribute to the early SVD seen in younger patients.At present, the technology to noninvasively study in vivo whether an implanted BHV in a human patient is undergoing rejection is not available. Thus, a small animal discordant xenotransplant model in young rodents (to match the young patient getting a pig/bovine/equine BHV) was developed to study whether the hypothesis that glutaraldehyde-fixed BHV undergo xenograft rejection had any merit. In this chapter, we describe our model and its merits and the results of our investigations. Our work provides clear evidence of xenograft rejection in glutaraldehyde-fixed tissue, and our small animal model offers an opportunity to study this process in detail.Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4 days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2 days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx).