Xenotransplantation of porcine and bovine islets without immunosuppression using uncoated alginate microspheres.

Uncoated spherical hydrogel microspheres (calcium alginate, nominal M(r) exclusion of > 600 kD) 800-900 microns in diameter were employed to prevent immune rejection of discordant islet xenografts isolated from pigs and cows. The islets were immobilized in the microspheres and injected into the peritoneum of 14 nonimmunosuppressed streptozotocin (STZ)-induced diabetic C57BL/6J mice. Four recipients received islet grafts from bovine calves, and 10 received islet grafts from pigs. In the control group of 15 diabetic mice implanted with nonencapsulated islets, 6 received i.p. porcine islets and 5 received i.p. bovine islets, whereas remaining 4 received porcine islets under the kidney capsule. Plasma glucose concentrations in recipients of the alginate-encapsulated islets promptly dropped from a preimplantation value of 498 +/- 47 (mean +/- SEM) to 142 +/- 6 (bovine) and 178 +/- 7 mg/dl (porcine) during the first wk. All the animals sustained these levels for at least 1 mo. Two mice implanted with bovine islets subsequently reverted to diabetes (plasma glucose > 250 mg/dl) at 43 days postimplantation. The remaining grafts maintained function for > 10 wk. In contrast, nonencapsulated islets failed to function, or sustained euglycemia for < 4 days. Mice receiving encapsulated islets showed a 23-38% gain in body weight during the first mo after implantation, compared with < 1% (P < 0.002) and 32% (P = 0.84) for the untreated diabetic (n = 6) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (> 10 wk) revealed viable islets, with well-granulated alpha, beta, and delta cells; the external surfaces of the microreactors were free of fibrotic overgrowth and exhibited only occasional host cell adherence. Uptake studies with IgG and thyroglobulin (M(r) of 669 kD) suggest that the microreactors were permeable to molecules with a molecular weight of up to > 600 kD (including the various proteins of the complement system, M(r) of 24-570 kD). Spheres implanted in the peritoneum after only 1 wk stained positive for both IgG and for the C3 component of complement. These findings suggest that prolonged survival of discordant xenografts of porcine and bovine islets in the STZ diabetic mouse model can be achieved with uncoated alginate microspheres that are permeable to IgG and complement. The question of whether similar results can be achieved with uncoated alginate microspheres in higher animals remains to be fully determined.

Biohybrid artificial pancreas. Long-term function of discordant islet xenografts in streptozotocin diabetic rats.

Long-term function of canine, bovine, and porcine islet xenografts implanted in streptozotocin-induced diabetic rats has been achieved by islet encapsulation within permselective acrylic membrane chambers. Intraperitoneal implants of 1 x 10(4) (n = 11) or 2 x 10(4) (n = 2) encapsulated canine islets reversed the diabetic state of the recipients within 24 hr, with plasma glucose levels dropping from a preimplantation level of 480 +/- 26 (mean +/- SEM) to 97 +/- 4 mg/dl during the first month. Chambers from 2 of the animals were removed, bisected, and reimplanted at 1 week and 2 months; both animals reverted to hyperglycemia (glucose, > 200 mg/dl) in < 2 weeks. The remaining implants maintained function for a mean time of 138 +/- 16 days, whereas the 2 animals that received the higher islet dose maintained function for > 260 days. Membranes containing 2 x 10(4) bovine (n = 6) or porcine (n = 10) islets also normalized glucose concentrations, with plasma glucose levels dropping from 468 +/- 61 to 91 +/- 10 (bovine) and 97 +/- 11 (porcine) mg/dl during the first month (vs. 94 +/- 3 mg/dl for nondiabetic control rats). Three of the latter implants were removed at 1 month. All 3 animals promptly reverted to diabetes. The 3-, 6-, 9-, and 12-month graft survival rates for the remaining animals were 100%, 100%, 60%, and 40%, and 100%, 75%, 50%, and 25%, respectively. The transplant recipients showed an approximately 38-54% gain in body weight during the first 100 days after implantation, compared with < 1% (P < 0.001) and 86% (P < 0.001) for the untreated diabetic (n = 5) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (1-20 months) revealed varying degrees of alpha-, beta-, and delta-cell granulation; the external membrane surfaces were generally free of fibrotic overgrowth and exhibited only occasional host cell adherence. Despite a problem of membrane breakage in long-term implants, these results suggest that prolonged survival of discordant transplants of porcine, bovine, and canine islets in diabetic rats can be achieved without immunosuppression.

Treatment of severe diabetes mellitus for more than one year using a vascularized hybrid artificial pancreas.

We report the successful application of a hybrid artificial pancreas device for the treatment of severe diabetes mellitus induced by total pancreatectomy in two dogs. Control of the blood sugar was achieved for more than 1 year in these two animals without any immunosuppressive therapy. Although exogenous insulin was required therapy. Although exogenous insulin was required during the latter part of the study period, removal of the devices resulted in a rapid increase in the fasting blood sugar levels and the exogenous insulin requirements (P < 0.001 versus weeks 1-52 in both dogs). Metabolic studies, postexplant in vitro studies, and histologic analyses confirmed islet cell survival and insulin production by the devices. This hybrid artificial pancreas has a clear clinical potential for islet cell transplantation without immunosuppression.

Transplantation of encapsulated canine islets into spontaneously diabetic BB/Wor rats without immunosuppression.

Extended survival of canine islet xenografts implanted in spontaneously diabetic BB/Wor rats has been achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes. Intraperitoneal implantation of the encapsulated islets reversed the diabetic state of the 10 recipients within 24 h. Plasma glucose levels declined from a preimplantation level of 459 +/- 30 to 102 +/- 14 mg/dl during the first 10 days. All of the animals sustained these levels for at least 1 month, and 2 animals for at least 2 and 8 months, respectively. To confirm that glucose homeostasis resulted from the encapsulated islet grafts, the implants were removed from 2 rats 1 month postimplantation, whereas a third was removed at 2 months. Hyperglycemia was observed immediately in all 3 animals, with glucose levels rising from 100 +/- 3 to 510 +/- 43 mg/dl within 1 day. In contrast, diabetic control rats (n = 4) receiving nonencapsulated islets became hyperglycemic in less than 1 week. The iv glucose tolerance test K value (decline in glucose levels, percent per min) at 10 days was 2.3 +/- 0.4 compared with 0.6 +/- 0.1 (P less than 0.005) and 3.1 +/- 0.1 (P less than 0.02) for untreated diabetic (n = 4) and normal control (n = 4) groups. Histological analyses and electron microscopy of long term functioning grafts revealed well preserved islets, with hormone-producing alpha-, beta-, and delta-cells; the membranes were generally free of fibrosis and host cell adherence. These results demonstrate that permselective artificial membranes can protect discordant islet xenografts from both graft rejection and autoimmune destruction for more than 1 month in an animal model that is similar in several respects to human type I diabetes.

Islet transplantation using an immunoprotective membrane in dogs that have undergone a pancreatectomy.

The use of a selectively permeable membrane to transplant nonsyngeneic tissue without accompanying immunosuppressive therapy has been investigated using two approaches. The first hybrid artificial pancreas is implanted as a vascular shunt in which blood circulates through the lumen of a tubular membrane. The islet tissue is distributed within a chamber surrounding the membrane enclosed by an acrylic housing. Studies with diabetic dogs that have had pancreatectomies have demonstrated that these devices could replace exogenous insulin therapy for at least 6 months in five animals. This report presents data on two of these dogs, demonstrating viability and function of the transplanted tissue after 1 year. As an alternative to the vascular device, islets sealed within cylindrical permselective membrane chambers have been implanted in the peritoneum. Preliminary data from three dogs indicate that the nonvascular implants can also regulate fasting glucose levels in the diabetic dog model.

Xenotransplantation of canine, bovine, and porcine islets in diabetic rats without immunosuppression.

Permselective acrylic membranes were employed to prevent immune rejection of discordant islet xenografts isolated from various large animals. Canine, porcine, and bovine islets were seeded into tubular diffusion chambers and transplanted into the peritoneum of 27 nonimmunosuppressed streptozotocin-induced diabetic Lewis rats. Six recipients received islet grafts from bovine calves, 7 received grafts from pigs, and 14 received grafts from dogs. Four of the latter were removed at 1 month. In the control group of 10 diabetic rats, 4 received nonencapsulated canine islets, 3 received nonencapsulated bovine islets, and 3 received nonencapsulated porcine islets. Recipients of encapsulated islets promptly dropped from a pretransplantation plasma glucose level of 487 +/- 36 (mean +/- SEM) to 84 +/- 2 (canine), 81 +/- 4 (bovine), and 81 +/- 3 mg/dl (porcine) during the first week. All of the animals sustained these levels for at least 1 month. One rat spontaneously reverted to diabetes at 54 days posttransplantation; 4 other rats became hyperglycemic (glucose, greater than 600 mg/dl) after membrane removal on day 30. The remaining 22 rats maintained fasting euglycemia for greater than 10 weeks. In contrast, rats that received nonencapsulated islets became hyperglycemic in less than 7 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 month for the canine and bovine encapsulated islet transplant group were 3.5 +/- 0.3 and 3.3 +/- 0.1 compared with 3.3 +/- 0.1 (P = 0.63) and 0.91 +/- 0.1 (P less than 0.0001) for normal (n = 4) and diabetic (n = 4) control groups. Morphologic studies of long-term functioning grafts (30-130 days) revealed well-preserved alpha, beta, and delta cells, with varying degrees of granulation. These results demonstrate that immune isolation of islet tissue using permselective artificial membranes can protect discordant islet xenografts from immune rejection in the absence of any immunosuppressive drugs.