Encapsulation of pig islets by alginate matrix to correct streptozotocin-induced diabetes in primates without immunosuppression

Allogeneic islet transplantation faces serious difficulties since organ shortage is recurrent and the lack of donors is aggravated by the fact that multiple pancreas donors are often necessary to treat one diabetic recipient. Another source of insulin-producing cells would therefore be of major interest and pigs represent a possible and serious option to provide such cells. However, pig-islet grafts could appear difficult due to the species barrier, but recent report seems to prove that pig islets function in primates for months. The main hurdle for cellular xenograft, however, remains the need for powerful and probables clinically inapplicable immunosuppression. We therefore focused our work on islet encapsulation in order to evaluate the possible use of pig cells without any immunosuppression. (i) This experimental work first evaluated the influence of pig age (young versus adult pigs) on islet size, collagen and vascular structures. Adult pig donors were selected since their pancreases are composed of a higher amount of large and well structured islets than those of young pigs. In addition, these large islets lend themselves to isolation and transplantation. Despite the adult age, the galactosyl epitope was still expressed on micro-vessels into the islets after the isolation procedure, thereby remaining a target for xenogeneic humoral response. (ii) After selection of the pig age, all the parameters involved in pancreas harvesting and pig-islets isolation procedures were investigated. Morphological screening of pig-pancreas donor prior to isolation, optimal blood exsanguination, warm ischaemic time <10 minutes and endotoxin content <30 Endotoxin Unit/mg in Liberase PI batches were significant determinants of successful pig islet isolation as assessed by multivariate analysis. (iii) Finally, a static method of pig-islet isolation was compared with the "gold standard" dynamic method. The static method seemed as efficient in term of islet yield as the dynamic method but needed less time, less technician assistance and provided a very pure isolation in a significantly lower volume. This method was therefore preferred and also successfully applied in humans. (iv) Prior to evaluation of the effect of encapsulation on pig islets in diabetic monkeys, the biocompatibility of the material used was first evaluated in vivo. The biocompatibility of encapsulated pig islets in a simple alginate bead was assessed in a non-diabetic primate model without immunosuppression. Long-term stability and biocompatibility for alginate-encapsulated pig islets were found up to 6 months after implantation under the kidney capsule in non-diabetic primates. A small proportion of these encapsulated pig islets not only survived up to 6 months but were also able to respond in vitro to a glucose+Forskolin challenge, thereby demonstrating an endocrine function. This in vivo experiment also allowed identification of important parameters for successful encapsulation: encapsulated pig islets need to be embedded in very pure alginate, cultivated for 18 or 24 hours in serum-free medium containing a concentration of 1.8mM of CaCl2, and transplanted with a minimum of 90% of well-formed capsules. A second in vivo study was carried out in a rat model in order to assess the impact of the implantation site on biocompatibility. In rats, the kidney subcapsular and subcutaneous spaces appeared more appropriate for encapsulated-pig-islet transplantation than the peritoneum. (v) Since long-term biocompatibility and even partial function were obtained in non-diabetic monkeys, it was therefore crucial to develop a suitable model of diabetes induction. In primates, a low dose (50 mg/kg) of streptozotocin (STZ) induced irreversible diabetes due to the selective destruction of more than 97% of insulin mass in the pancreas, without any side-effects on liver and renal function. (vi) After induction of diabetes by STZ in primates, the ability of encapsulated pig islets to reverse diabetes after transplantation under the kidney capsule was evaluated. Microencapsulated adult pig islets provided a short-term and partial correction of hyperglycaemia in all animals, but the multi-layer graft under the kidney capsule demonstrated the limitation of this model probably due to insufficient oxygenation of the encapsulated b-cells. (vii) We therefore developed a mono-layer cellular device (MCD) in order to transplant diabetic primates in the subcutaneous space, which is also more clinically applicable. Although the results are preliminary in four primates, we clearly demonstrated that this strategy could be an appropriate solution, since this mono-layer device containing pig-pancreatic cells controls the diabetic status for at least 4 months without any immunosuppression. These results need obviously to be confirmed in a larger cohort of primates, but these results are of overwhelming importance since they were obtained without immunosuppression. This model could therefore not only be applied to diabetic patients who need combined renal and islet transplant but also to young diabetic patients who do not yet present major side-effects of diabetesThèse de doctorat en sciences biomédicales (transplantation) (SBIM 3)--UCL, 200

Islet cell xenotransplantation: update on recent progress and future perspectives.

Allogeneic islet transplantation faces difficulties because (i)organ shortage is recurrent; (ii) several pancreas donors are often needed to treat one diabetic recipient; and (iii) the intrahepatic site of islet implantation may not be the most appropriate site. Another source of insulin-producing cells, therefore, would be of major interest, and pigs represent a possible and serious source for obtaining such cells. Pig islet grafts may appear difficult because of the species barrier, but recent reports demonstrate that pig islets may function in primates for at least 6 months. Pig islet xenotransplantation, however, must still overcome several hurdles prior to becoming clinically applicable. The actual consensus is to produce more preclinical data in the pig-to-primate model as a necessary requirement to envisage any pig-to-human transplantation of islets; therefore, a summary of the actual acquired knowledge of pig islet transplantation in primates seemed useful

Pig islet for xenotransplantation in human structural and physiological compatibility for human clinical application.

Allogeneic islet transplantation has proven difficult because organ shortages are recurrent, several pancreas donors are often needed to treat one diabetic recipient, and the intrahepatic site of islet implantation may not be the most appropriate site. Thus, another source of insulin-producing cells would be beneficial; and pigs represent a possible and viable source for obtaining such cells. Although the use of pig islet grafts appears to be difficult because of the species barrier, recent reports demonstrated that pig islet xenotransplantation can overcome the immunological barrier following strong immunosuppression and function successfully in primates for at least 6 months. Before becoming clinically applicable, however, pig islet xenotransplantation must still overcome the structural and physiological incompatibility between pig donor and human recipient. Researchers agree that it is necessary to produce more preclinical data in the pig-to-primate model before any pig-to-human transplantation of islets can be considered. Therefore, in this review, we provide a summary of the present state of knowledge about pig and human islet compatibility

Islets Xenotransplantation

Allogeneic islet transplantation faces serious difficulties since organ shortage is recurrent and the lack of donors is aggravated by the fact that several pancreas donors are often needed to treat one diabetic recipient. Another source of insulin-producing cells would therefore be of major interest and pigs represent a possible and serious way of obtaining such cells. Pig islet grafts may appear difficult due to the species barrier, but recent reports demonstrate that pig islets may function in primates for at least 6 months. However, pig islet xenotransplantation, in the context of clinical application, still has to overcome several hurdles such as selection of pig islet donors (germ-free conditions), maturity, isolation and function of islets, possible xenozoonosis, as well as a strong immunosuppression of the recipient. The choice between clinically applicable immune regimens and/or encapsulation is also discussed. Recently acquired knowledge of islet transplantation is summarized in this manuscript

Correction d'un diabète de type I chez le primate par greffe d'îlots pancréatiques porcins encapsulés.

The allogeneic transplantation of pancreatic islet cells into diabetic patients is severely restricted by the recurrent lack of organs. To the lack of donors is added the fact that several pancreas donors are often needed in order to treat a single diabetic recipient. Another source of cells that can produce insulin would therefore be of major interest. The pig constitutes a possible option to be taken seriously. Nonetheless, grafting pig islets might prove difficult because of the species barrier. The principal obstacle to the xenotransplantation of cells remains the necessity for powerful immunosuppression, probably inapplicable in man. Therefore, we have centred our work around the encapsulation of islets in order to evaluate the possibility of using pig cells without immunosuppression. (1) This experimental work first evaluated the influence of the age of the pig (young versus adult pigs) on the size of the islets, the collagen and the vascular structures. Adult donor pigs were selected. (Dufrane et al., Pancreas 30(2), 2005). (2) After deciding on the age, all the parameters involved in the removal of the pancreas and the procedures of isolating the pig islets were scrutinized by multivariable analysis. (Dufrane et al., Xenotransplantation 13(3), 2006). (3) Before evaluating the effect of encapsulation on the pig islets in diabetic monkeys, the biocompatibility of an alginate membrane was first evaluated in a non-diabetic primate model without immunosuppression. The long term stability and biocompatibility of pig islets encapsulated in alginate have been confirmed for up to six months after implantation under the renal capsule. A small proportion of these encapsulated pig islets not only survived for six months but remained capable of reacting in vitro to stimulation with glucose + forskolin, thus demonstrating preserved endocrine function. This in vivo experience has enabled us to identify the most important parameters for successful encapsulation. (Dufrane et al., Transplantation 81(9), 2006; Dufrane et al., Biomaterials 27(17), 2005). We then (4) investigated the most appropriate means of inducing irreversible diabetes in primates: a low dose (50 mg/kg) of streptozotocine (STZ) permanently destroys more than 97% of the insulin-producing cells of the pancreas, without any side effects on hepatic or renal function. (Dufrane et al., Transplantation 81(1), 2006 (5) After induction of diabetes by STZ in primates, the capacity of encapsulated pig islets to control diabetes after transplantation was evaluated both under the renal capsule and in the form of a graft of a monolayer of cells in the subcutaneous space. The second technique enabled the diabetic status to be controlled for at least six months without any immunosuppression in four primates. This result is unique since only major immunosuppressant regimes have so far brought comparable results

Macro- or microencapsulation of pig islets to cure type 1 diabetes.

Although allogeneic islet transplantation can successfully cure type 1 diabetes, it has limited applicability. For example, organs are in short supply; several human pancreas donors are often needed to treat one diabetic recipient; the intrahepatic site may not be the most appropriate site for islet implantation; and immunosuppressive regimens, which are associated with side effects, are often required to prolong survival of the islet graft. An alternative source of insulin-producing cells would therefore be of major interest. Pigs represent a possible alternative source of beta cells. Grafting of pig islets may appear difficult because of the immunologic species barrier, but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression. Therefore, a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation. Although several groups have shown that encapsulated pig islets are functional in small-animal models, less is known about the use of bioartificial pancreases in large-animal models. In this review, we summarize current knowledge of encapsulated pig islets, to determine obstacles to implantation in humans and possible solutions to overcome these obstacles

Pig islets for clinical islet xenotransplantation

PURPOSE OF REVIEW: Allogeneic islet transplantation faces difficulties because organ shortage is recurrent; several pancreas donors are often needed to treat one diabetic recipient; and the intrahepatic site of islet implantation may not be the most appropriate one. Another source of insulin-producing cells, therefore, would be of major interest, and pigs represent a possible and serious source for obtaining such cells. RECENT FINDINGS: Pig islet grafts may appear difficult because of the species barrier, but recent studies demonstrate that pig islets may function in diabetic primates for at least 6 months. SUMMARY: Pig islet xenotransplantation, however, must still overcome the selection of a suitable pig donor to translate preclinical findings into clinical applications. This review summarizes the actual acquired knowledge of pig islet transplantation in primates to select the 'ideal' pig donor

Pig islet xenotransplantation

Allogeneic islet transplantation faces serious difficulties since organ shortage is recurrent and the lack of donors is aggravated by the fact that several pancreas donors are often needed to treat one diabetic recipient. Another source of insulin-producing cells would therefore be of major interest and pigs represent a possible and serious way of obtaining such cells. Pig islet grafts may appear difficult due to the species barrier, but recent reports demonstrate that pig islets may function in primates for at least six months. However, pig islet xenotransplantation, in the context of clinical application, still has to overcome several hurdles such as selection of pig islet donors (germfree conditions), maturity, isolation and function of islets, possible xenozoonosis, as weil as a strong immunosuppression of the recipient. The choice between clinically applicable immune regimens and/or encapsulation is also discussed. Recently acquired knowledge of islet transplantation is summarized in this chapter