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.

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.

Investigation of the regeneration potential of the recurrent laryngeal nerve (RLN) after compression injury, using neuromonitoring.

INTRODUCTION: The aim of this study was to investigate the regeneration potential of RLN after the compression of the nerve, without disrupting its continuity, using neuromonitoring. METHODS: In the first operation, the RLN and nervus vagus of adult Goettingen minipigs were dissected free, and the neuromonitoring parameters (amplitude, threshold and lag time of signal) were measured. Injury of the RLN was induced using a "bulldog" clamp. When the signal was no longer detectable, after the 15 min regeneration phase, the operation was finished. The neuromonitoring studies (see above) were repeated in a second operation 6 months later. RESULTS: (1) After the first operation, acute clamping of the RLN led to a reduction in the amplitude of the neuromonitoring signal; the lag time and the threshold of signal remained. Complete restitution of the signal was observed during the first regeneration phase. Repeated clamping led to complete disappearance of the signal. (2) During the second operation, i.e., after 6 months of regeneration, the neuromonitoring signals of both RLN and nervus vagus were detected in 93% of the GMP. No statistical differences (p = 0.17) were noticed between the amplitude of the RLN before the nerve injury (first operation) and after nerve regeneration (second operation). A significant increase in the lag time (p < 0.0005) was shown for both RLN and nervus vagus. CONCLUSIONS: The acute compression of RLN can only be detected by observing the amplitude of the neuromonitoring signal. Restitutio ad integrum is possible after a short clamping period but it is important to preserve the RLN continuity.

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.

Preoperative evaluation of microencapsulated human parathyroid tissue aids selection of the optimal bioartificial graft for human parathyroid allotransplantation.

Allotransplantation of microencapsulated parathyroid tissue is a promising approach to the treatment of permanent hypoparathyroidism. Preoperative assessment of the quality of microencapsulated parathyroid tissue could facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation. Parathyroid tissue from patients with secondary hyperparathyroidism (n = 15) was processed mechanically or enzymatically (collagenase type II). Tissue particles and single cells/cell clusters were routinely microencapsulated with amitogenic Ba(2+) alginate. Parathyroid secretion dynamics in response to stimulation of nonencapsulated and microencapsulated parathyroid tissue with Ca(2+) were evaluated in a perifusion system. The stability of the different types of microcapsule was assessed using an osmotic pressure test. Mechanical cutting of parathyroid tissue led to peripheral necrosis of tissue particles and impaired their vitality. Collagenase digestion, in contrast, resulted in single cells and cell clusters without peripheral necrosis. The quality of microencapsulation of single cells/cell clusters was significantly better than that of tissue particles (deformed and imperfect capsules). Microencapsulation itself did not decrease cell vitality. Nonencapsulated and microencapsulated tissue particles and single cells/cell clusters from different donors maintained their own levels of response to stimulation with low Ca(2+). Microcapsules containing tissue particles showed poor stability compared with those containing single cells/cell clusters. Preoperative evaluation of microencapsulated parathyroid tissue can disclose differences in vitality and function and thus facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation.

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.