Beta-O2 Technologies (Israel)

Xenotransplantation using pig cells, tissues or organs may be associated with the transmission of porcine zoonotic micro-organisms. Hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine endogenous retroviruses (PERVs) are potentially zoonotic micro-organisms which do not show clinical symptoms in pigs and which are due to the low expression level difficult to detect. Göttingen Minipigs (GöMP) are often used for biomedical investigations and they are well characterized concerning the presence of numerous bacteria, fungi, viruses and parasites and therefore may be used for islet cell transplantation. Islet cells derived from three GöMP were transplanted into four healthy, non-diabetic cynomolgus monkeys using a macroencapsulation device. PCR, nested PCR, real-time PCR, real-time RT-PCR and Western blot analyses were used to estimate the presence of PERV, PCMV and HEV in the donors and recipients. Using sensitive detection methods, no HEV was found in the donor pigs and in the pig islet cell preparations. Antibodies against PERV, PCMV and HEV were not found in all cynomolgus monkeys with exception of one monkey showing an immune response against HEV. Using real-time PCR, no PCMV and HEV were found in the sera of all monkeys. Although the donor islet cells and the recipients were negative for HEV using PCR and Western blot analysis, in one recipient, antibodies against HEV were found, indicating infection in a single case. All recipients were negative for antibodies against PERV, and all were negative for PCMV, indicating absence of infection. As HEV was not detected in the donor pig before transplantation, a more complex and regular screening of the animals using highly sensitive methods is required to avoid virus transmission.

At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets of Langerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane (e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, especially when using large retrievable macro-capsules implanted in poorly oxygenated sites like the subcutis. Notably, islets can survive under low oxygen tension and are physiologically active at > 40 Torr. Surprisingly, when densely crowded, islets are fully functional under hyperoxic pressure of up to 500 Torr (> 300% of atmospheric oxygen tension). The review also addresses an additional category of requirements for optimal performance of transplanted islets, named auxiliary technologies. These include control of inflammation, apoptosis, angiogenesis, and the intra-capsular environment. The review highlights that curing diabetes with a functional bio-artificial pancreas requires optimizing all of these aspects, and that significant advances have already been made in many of them.

Introduction: Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications by reconstitution of endogenous insulin secretion. However, gradual loss in graft function over time due to insufficient oxygenation and inflammatory reactions, and chronic need for immunosuppression limit the outcome and the indication for islet transplantation. Methods: Here we present the approach of utilization of a macrochamber specially engineered for islet transplantation. The subcutaneous implantable device allows for controlled and adequate oxygen supply and provides immunological protection. In an isogeneic (n=16) and allogeneic (n=7) rodent model, pancreatic islets were embedded in alginate, integrated into the chamber-system and transplanted into Streptozotocin-induced diabetic rats. In addition, islets were pre-treated with growth hormone-releasing hormone (GHRH) agonist, JI-36. Prior to transplantation, islets were characterized for morphology and function by glucose stimulated insulin release, determination of oxygen consumption rate and immunohistochemistry. During the follow-up period of up to 90 days, blood glucose levels were measured daily and animals were subjected to an intravenous glucose tolerance test. The islet graft was removed at the end of the observation period to ensure recurrence of diabetes and enabling post-explantation assessment of the islet graft and the chamber surrounding tissue. Results: The subcutaneous chamber containing the islet graft normalized blood glucose in diabetic rodents for up to 3 months.Figure: [Metablic follow up of transplanted rodents]Sufficient graft function depended on oxygen supply. Pre-treatment with GHRH agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing in β-cell insulin reserve thereby allowing for a significant reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Despite complete avoidance of immunosuppression in the allogeneic transplantation model, no signs of rejection were observed, indicating a sufficient immunoisolation of the islet graft by the chamber system. Conclusion: A more efficient use of donor islets could facilitate the widespread use of islet transplantation as a treatment approach for type 1 diabetes. We investigated the combination of GHRH agonism with ideal oxygenation using a bioartificial macrochamber. Encapsulated islets are maintained within a device, which creates a sufficient immune barrier and allows for adequate oxygen supply to the islet graft. Together, a new strategy capable of restoring and maintaining long-term euglycemia in diabetic models is thereby presented. Consequently, this system opens up a novel strategy for β-cell replacement therapy without the need for immunosuppression and may even provide a promising avenue for future approaches to xenotransplantation.

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing β-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

Immunoisolation of pancreatic islets interrupts their vascular connections and results in severe cell hypoxia and dysfunction. This process is believed to be the major obstacle to a successful cure of diabetes by implantation of bioartificial pancreas. Here we describe a new technology for microalga-based, photosynthetic oxygen supply to encapsulated islets, in which a thermophylic strain of the unicellular alga Chlorella was used as a natural photosynthetic oxygen generator. Following determinations of the optimal number of alga cells required for compensation of islet respiration, an appropriate number of islets and algae were co-encapsulated in alginate and perifused with oxygen-free medium at increasing glucose concentrations. No insulin response to glucose was obtained in islets alone, or upon inactivation of photosynthesis by darkness. However, under illumination, photosynthetic- dependent oxygen generation induced higher glucose-stimulated insulin response when compared to normoxic perifusion. Such photosynthetic oxygen generation may have a potential application in development of various bioartificial tissues, in particular the endocrine pancreas.