Reversal of diabetes in dogs by transplantation of pure cryopreserved islets

We have autotransplanted highly purified cryopreserved canine islets into pancreatectomized dogs. Islets were frozen by slow cooling (0.25 degrees C/min) to -40 degrees C, stored at -196 degrees C and thawed rapidly (200 degrees C/min). A total of 7868 +/- 665 (means +/- SE) cryopreserved islets/kg body weight were implanted to the spleen (n = 7) by venous reflux, and seven other control dogs received 6811 +/- 653 fresh islets/kg (P versus cryo., NS). Fasting plasma glucose [( PG] mg/dl, +/- SEM) and intravenous glucose tolerance were determined before and at 1 and 3 months postimplantation. Six dogs that received cryopreserved islets and all seven control animals promptly became normoglycemic, with PGs of 133 +/- 23 and 110 +/- 4, respectively, at 1 week postimplantation (NS). During follow-up one normoglycemic animal from each group died due to small bowel volvulus and one recipient of fresh islets became diabetic at 14 days. The remaining dogs remained normoglycemic with PGs of 89 +/- 3 and 92 +/- 3 in dogs receiving cryopreserved and fresh islets, respectively, at 3 months postimplantation (NS). Mean K value (decline of glucose, %/min +/- SEM) at ivGTT for all dogs was 2.5 +/- 0.3 preoperatively. At 1 and 3 months postimplantation, the K values of dogs receiving cryopreserved islets were 1.3 +/- 0.1 and 1.4 +/- 0.2, respectively, compared with 1.3 +/- 0.2 and 2.0 +/- 0.2 for control dogs (NS). These data demonstrate prompt and sustained function of a defined quantity of frozen-thawed purified islets in a large mammal. Cryopreservation is an effective method for the long-term storage of purified islet tissue.     

Reversal of diabetes in BB rats by transplantation of encapsulated pancreatic islets

Prolonged survival of pancreatic islet allografts implanted in diabetic BB rats was achieved by encapsulation of individual islets in a protective biocompatible alginate-polylysine-alginate membrane without immunosuppression. Intraperitoneal transplantation of the encapsulated islets reversed the diabetic state of the recipients within 3 days and maintained normoglycemia for 190 days. Normal body weight and urine volume were maintained during this period, and no cataracts were detected in the transplant recipients. In contrast, control rats receiving transplants of unencapsulated islets experienced normoglycemia for less than 2 wk. These results demonstrated that microencapsulation can protect allografted islets from both graft rejection and autoimmune destruction without immunosuppression in an animal model that mimics human insulin-dependent diabetes.     

Microencapsulation of cryopreserved islets for transplantation

Purpose: The development of islet banks and of techniques to immunoisolate islets by encapsulation has the potential to overcome the major barriers to the routine use of islet transplants as an alternative to insulin therapy in diabetic patients.Aim: The purpose of this study was to determine the in vitro function of islets stored by cryopreservation prior to microencapsulation.Method: Islets were isolated from the pancreata of Sprague-Dawley rats and stored frozen at −80°C in the presence of cryoprotectants for 1 month. After thawing and overnight culture, they were tested either unencapsulated or after microencapsulation with poly-l-lysine-alginate membrane using an air-jet syringe pump. Islets were tested for response to glucose stimulation using a perifusion procedure.Results: In the unencapsulated islets, the mean ± SEM rate of insulin secretion increased from a basal value of 832 ± 34 to a peak of 1448 ± 138 pg/6 islets/min, p < .01, n = 5) within 10 min of raising the glucose concentration from 3.3 mM (60 mg/dl) to 16.7 mM (300 mg/dl). Similarly, insulin secretion in the microencapsulated islets increased from a basal value of 649 ± 6 to a peak of 1184 ± 100, p < .01, with restoration of basal rate on return of islets to basal perifusion. The characteristics of the response to glucose stimulation of these cryopreserved islets were comparable with those seen in freshly isolated islets.Conclusion: Islets cryopreserved for 1 month retained viability after thawing and microencapsulation.     

Diurnal insulin and glucose profiles following transplantation of fresh and cryopreserved canine pancreatic islets

Dispersed islet autotransplants (fresh and cryopreserved) can produce normal fasting insulin and glucose concentrations. Following an intravenous glucose challenge, however, the insulin response is impaired and glucose clearance delayed. This study examines the diurnal insulin and glucose response to feeding in recipients of fresh islet autografts (n = 6), recipients of cryopreserved islet autografts (n = 3), normal unoperated controls (n = 10) and insulin treated pancreatectomized diabetic dogs (n = 7). Mean fasting insulin and glucose levels were similar in all groups. After feeding, insulin profiles in both transplant groups were near normal while significant hyperinsulinaemia occurred in insulin treated diabetic dogs. Mild postprandial carbohydrate intolerance was observed in transplant recipients but glycosylated haemoglobin levels were similar (mean(s.d.] to normals (6.88(0.86) per cent, 6.86(0.5) per cent respectively). Dispersed islet autotransplantation with portal insulin delivery produces near normal insulin and glucose profiles suggesting that this may represent a suitable treatment for insulin dependent diabetes.     

Reversal of diabetes in pancreatectomized pigs after transplantation of neonatal porcine islets

Neonatal porcine islets (NPIs) are able to grow and to reverse hyperglycemia after transplantation in immunoincompetent mice. The aim of this study was to demonstrate the feasibility of allogeneic NPI grafts to achieve normoglycemia in a pancreatectomized diabetic pig. NPIs were isolated from pancreases of 1- to 3-day-old pigs, cultured, and then transplanted via the portal vein into the liver of totally pancreatectomized pigs (mean body weight, 20.8 kg). Each pig received NPIs consisting of 3.1 +/- 0.3 x 10(6) beta-cells/kg (12,476 +/- 1,146 islet equivalent/kg). The six pigs that were given cyclosporine and sirolimus achieved normoglycemia by day 14 without insulin therapy. Three pigs died of surgical complications shortly after transplantation, whereas the other three remained insulin independent up to day 69. Of seven nonimmunosuppressed recipients, four pigs became normoglycemic by day 14 without insulin treatment, with two of the animals remaining normoglycemic long term. Well-preserved insulin-positive cells were found in the graft at the end of follow-up with a significant increase in insulin content in long-term survivors of both groups. This study demonstrates for the first time that allogeneic NPIs can reverse hyperglycemia in totally pancreatectomized diabetic pigs.     

微重力培养提高冻存大鼠胰岛移植的效果

目的观察大鼠胰岛冻存后经三维微重力细胞旋转培养系统(RCCS)培养后移植给受者能否明显提高胰岛移植的效果。方法将分离纯化的大鼠胰岛分为3组。实验1组:即将胰岛在RCCS中培养3d后,悬浮于4℃低温保存液(HTS)中,放置30min,然后进行冻存步骤;冻存1个月后进行复苏,继续在RCCS中培养30d;实验2组:新鲜大鼠胰岛在RCCS中培养30d;对照组:大鼠胰岛在冻存前后均在普通培养基中培养,培养步骤和时间同实验1组。上述3组胰岛复苏后再按各组应用的培养体系培养7d,然后分别以2000IEQ冻存或新鲜胰岛移植入糖尿病大鼠体内,并观察10周。结果每个胰腺纯化后最多收获胰岛1058.47IEQ,最少411.88IEQ,平均(826.95±93.42)IEQ。实验1组、实验2组的胰岛收获率、细胞内胰岛素和DNA含量以及胰岛素刺激指数均高于对照组(P<0.05);实验2组和实验1组的2000IEQ新鲜胰岛或冻存胰岛在移植后1周内可100%纠正糖尿病。结论大鼠胰岛冻存前后经微重力培养后移植可以明显提高1型糖尿病大鼠的治疗效果。     

Reversal of hyperglycemia in mice after subcutaneous transplantation of macroencapsulated islets

BACKGROUND: Macroencapsulated islets can reverse hyperglycemia in diabetic animals when transplanted i.p. or into the fat pad. The s.c. space is an attractive site for such transplantation because macrocapsules can be implanted with local anesthesia and be easily removed or reloaded with fresh islets. METHODS: Immunoprotective 20 microl ported devices were transplanted under the skin of Streptozocin-diabetic nude mice. Devices were loaded with 1200 rat islets in culture medium or in alginate. Empty devices were implanted for 2 weeks and then loaded with islets. Normal mice and mice with islets transplanted under the renal capsule or under the skin were used as controls. Seven weeks after transplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed, followed by implant removal. RESULTS: Three weeks after transplantation, normal blood glucose levels were observed in all animals. Compared with those of normal controls, IPGTTs showed accelerated blood glucose clearance in mice transplanted with islets either within devices or beneath the kidney capsule. Fasted transplanted mice were hypoglycemic before glucose injection and 2 hr later. After removal of the implants, all recipient mice returned to hyperglycemia. Histological evaluation revealed viable islet cells and a network of close vascular structures outside the devices. CONCLUSIONS: Macroencapsulated islets transplanted into the s.c. space were able to survive and regulate blood glucose levels in mice. The observed differences in glucose metabolism between normal and transplanted mice may be attributed to the site of transplantation and to the use of rat islets, which have a different set point for glucose induced insulin release.     

Reversal of diabetes by pancreatic islet transplantation into a subcutaneous, neovascularized device

BACKGROUND: Transplantation of pancreatic islets for the treatment of type 1 diabetes allows for physiologic glycemic control and insulin-independence when sufficient islets are implanted via the portal vein into the liver. Intrahepatic islet implantation requires specific infrastructure and expertise, and risks inherent to the procedure include bleeding, thrombosis, and elevation of portal pressure. Additionally, the relatively higher drug metabolite concentrations in the liver may contribute to the delayed loss of graft function of recent clinical trials. Identification of alternative implantation sites using biocompatible devices may be of assistance improving graft outcome. A desirable bioartificial pancreas should be easy to implant, biopsy, and retrieve, while allowing for sustained graft function. The subcutaneous (SC) site may require a minimally invasive procedure performed under local anesthesia, but its use has been hampered so far by lack of early vascularization, induction of local inflammation, and mechanical stress on the graft. METHODS: Chemically diabetic rats received syngeneic islets into the liver or SC into a novel biocompatible device consisting of a cylindrical stainless-steel mesh. The device was implanted 40 days prior to islet transplantation to allow embedding by connective tissue and neovascularization. Reversal of diabetes and glycemic control was monitored after islet transplantation. RESULTS: Syngeneic islets transplanted into a SC, neovascularized device restored euglycemia and sustained function long-term. Removal of graft-bearing devices resulted in hyperglycemia. Explanted grafts showed preserved islets and intense vascular networks. CONCLUSIONS: Ease of implantation, biocompatibility, and ability to maintain long-term graft function support the potential of our implantable device for cellular-based reparative therapies.     

Reversal of mesangial enlargement in rats with long-standing diabetes by whole pancreas transplantation

An important unanswered question about clinical use of pancreas transplantation is: can pancreas transplants reverse or, at least, stabilize well-established lesions of insulin-dependent diabetes mellitus (IDDM)? To answer this question, we performed whole pancreas transplantations in 190 highly inbred rats 6, 9, 12, 15, 18, and 21 mo after induction of diabetes mellitus (DM) with alloxan. We then studied the effect on renal mesangial enlargement (ME) for 24 mo after onset of DM by a quantitative morphologic technique in which camera lucida tracings of the mesangium were made at X 1250 and were analyzed using an electronic planimeter connected to a calculator/computer. A pretransplant kidney biopsy was obtained so that the rats served as their own controls. In addition, studies were performed for 28 mo in 57 untreated diabetic controls and in 55 nondiabetic controls. Monthly metabolic studies showed that whole pancreas transplantation maintained very tight, lifelong metabolic control of diabetes. Kidney sections obtained for 2 yr from diabetic controls and for 21 mo from diabetic rats before transplantation showed highly significant increases in total mesangial area, nuclear-free mesangial area, and percentage of glomerular area occupied by nuclear-free mesangial area. Pancreas transplantation consistently produced a highly significant reversal of well-established ME, regardless of when it was performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced recovery of cryopreserved islets using SIS

Although cryopreservation of pancreatic islets would add flexibility to transplantation, the recoveries are only 60% to 90% and function is decreased. Islets are multicellular structures approximately 50 to 250 microm in diameter organized into a network of cells and vascular channels. Due to this complexity, islets are more susceptible to damage during cryopreservation than an individual cell. This study investigated porcine small intestinal submucosa (SIS) as a matrix to support islets recovery and function post-thaw. Groups of frozen/thawed human islets (150 IE/condition; n = 4 preparations) were cultured for 5 weeks in plates containing noncoated Biopore membrane inserts alone or inserts covered with SIS. Islets were placed directly on the insert post-thaw (SIS(1)), or cultured overnight in standard plates, washed, and then transferred to the SIS (SIS(2)). Function was assessed by determining glucose-stimulated release of insulin, which was measured by radioimmunoassay. Analysis of basal insulin secretion showed time and treatment to be significantly different (P =.0043 and P =.0123, respectively) but without an interaction (P >.05). The two SIS treatments were not significantly different (P >.05); however, both SIS(1) and SIS(2) were significantly different from controls (P =.0108 and P =.0420, respectively). Similar results were obtained for stimulation indices; time and treatment were significantly different (P =.0161 and P =.0264, respectively) but not an interaction (P >.05). The two SIS treatments were not significantly different (P =.05); however, both SIS(1) and SIS(2) differed from controls (P =.0248 and P =.0407, respectively). The results indicate that SIS enables frozen-thawed islets to exhibit superior post-thaw function compared with a non-SIS-supported condition.     

高压氧微重力培养提高冻存胰岛质量和移植效果的实验研究

目的 探讨胰岛冻存前后经高压氧细胞旋转培养系统(HORCCS)培养后移植入糖尿病大鼠能否提高胰岛移植的效果.方法 将分离纯化的大鼠胰岛分为:A.体外实验组:将各组大鼠胰岛经HORCCS培养或普通培养30 d,检测细胞内DNA和胰岛素含量,胰岛存活率,胰岛素分泌水平.B.胰岛移植实验组:将各组大鼠胰岛经HORCCS培养或普通培养7d,然后移植,观察移植受体血糖和胰岛素水平.电镜观察各胰岛移植实验组中培养7 d时的胰岛的超微结构改变.结果 经高压氧 RCCS培养14 d的胰岛存活率及胰岛素分泌水平高于普通培养组(P<0.05).移植了经HORCCS培养的胰岛后,受体血糖在移植后2周即恢复为正常值,并维持到移植后10周.全部受体维持正常血糖耐受曲线.电镜下可见经HORCCS培养后,冻存复苏胰岛表面形成微小的孔道.结论 胰岛冻存前后经高压氧RCCS培养后可以建立营养输送管道,不但有利于氧和营养物质的运输,更有利于胰岛内细胞的均匀一致的冻存,从而减少冻存对胰岛的损害,提高胰岛的分泌活性和成活率.