Isolation of porcine pancreatic islets for xenotransplantation.

This chapter deals with a technique for isolating intact islets of Langerhans from the pig pancreas based on our experience performing approximately 750 isolations. The procedure we describe involves identification of an optimal donor pancreas, purification and in vitro culture of islets, diabetes induction in recipients, and transplantation of islets and their immunomodulation. Besides the sophistication of the technical equipment employed, the major factors influencing the isolation outcome are the pig breed, the number and morphology of the islets in the donor pancreas, the quality of the collagenase/neutral protease, and the skill of the team members.

Goettingen Minipigs (GMP): Comparison of Two Different Models for Inducing Diabetes.

PURPOSE: Preclinical experiments on large animals are indispensable for evaluating the effectiveness of diabetes therapies. Miniature swine are well suited for such studies due to their physiological and pathophysiological responses. METHODS: We compare two methods for inducing diabetes in Goettingen minipigs (GMP), in five with the beta cell toxin streptozotocin (STZ) and in five other GMP by total pancreatectomy (PE). Glucose homeostasis was assessed with the intravenous glucose-tolerance test (IVGTT) and continual monitoring of interstitial glucose levels. At conclusion of the observation period, the pancreata were examined histologically. Three non-diabetic GMP served as control group. RESULTS: The IVGTT revealed markedly diabetic profiles in both GMP groups. STZ-GMP were found to harbor residual C-peptides and scattered insulin-positive cells in the pancreas. PE-GMP survived the total pancreatectomy only with intensive postoperative care. CONCLUSIONS: Although both methods reliably induced diabetes in GMP, the PE-GMP clearly had more health problems and required a greater expenditure of time and resources. The PE-GMP model, however, was better at eliminating endogenous insulin and C-peptide than the STZ-GMP model.

Glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function.

OBJECTIVE: The insulinotropic action of the incretin glucose-dependent insulinotropic polypeptide (GIP) is impaired in type 2 diabetes, while the effect of glucagon-like peptide-1 (GLP-1) is preserved. To evaluate the role of impaired GIP function in glucose homeostasis and development of the endocrine pancreas in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPR(dn)) in pancreatic islets. RESEARCH DESIGN AND METHODS: GIPR(dn) transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the different endocrine islet cell populations were performed, and beta-cell proliferation and apoptosis were quantified to characterize this novel animal model. RESULTS: Eleven-week-old GIPR(dn) transgenic pigs exhibited significantly reduced oral glucose tolerance due to delayed insulin secretion, whereas intravenous glucose tolerance and pancreatic beta-cell mass were not different from controls. The insulinotropic effect of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in GIPR(dn) transgenic versus control pigs. With increasing age, glucose control deteriorated in GIPR(dn) transgenic pigs, as shown by reduced oral and intravenous glucose tolerance due to impaired insulin secretion. Importantly, beta-cell proliferation was reduced by 60% in 11-week-old GIPR(dn) transgenic pigs, leading to a reduction of beta-cell mass by 35% and 58% in 5-month-old and 1- to 1.4-year-old transgenic pigs compared with age-matched controls, respectively. CONCLUSIONS: The first large animal model with impaired incretin function demonstrates an essential role of GIP for insulin secretion, proliferation of beta-cells, and physiological expansion of beta-cell mass.

No transmission of porcine endogenous retroviruses (PERVs) in a long-term pig to rat xenotransplantation model and no infection of immunosuppressed rats.

BACKGROUND: Xenotransplantation from pig to humans may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs) that are present in the genome of all pigs and that infect human cells in vitro. However, it remains unclear whether PERVs infect transplant recipients in vivo and, if so, whether they are pathogenic. It is therefore essential to perform in vivo infection studies in animal models. MATERIAL/METHODS: To study PERV transmission in rats, rat primary cells and cell lines were treated in vitro with virus from different sources. Based on the assumption that susceptible cell lineages not yet tested in vitro could be present in the animal, PERV was inoculated into naïve and immunosuppressed animals. To investigate PERV transmission in a long-term exposure experiment, sera from animals grafted with pig Langerhans islet cells were tested in a Western blot assay for antibodies against PERVs. The animals were treated with streptozotocin to induce diabetes and microencapsulated and non-microencapsulated pig islet cells were applied without immunosuppression. RESULTS: No productive infection of a few selected rat primary cells or cell lines was observed in vitro. PERV-specific antibodies were found in none of the animals and no integration of PERV into rat cells of different organs was observed, indicating that infection had not occurred. CONCLUSIONS: This report demonstrates a lack of infection of rats in vivo even during immunosuppression or long-term exposure (up to 460 days) to a functioning xenotransplant. This report also shows that rats possibly due to a low receptor concentration on their cells are not a suitable animal model to study PERV transmission in vivo.

Interaction of polyelectrolytes and their composites with living cells.

Since the layer-wise polyelectrolyte deposition offers the opportunity to modify surfaces for biomedical applications, interactions and toxicity between polyelectrolytes and living cells become interesting. The aim of the present work is to determine the different factors such as contact area, charge, and transplantation site that influence the cell reaction to a specific polymer. We found that toxicity is influenced by all these factors and cannot be tested easily in a model.