A recently described method for isolation of islets of Langerhans from the human pancreas has been adapted to the cynomolgus monkey pancreas. The mean yield of islets obtained from 13 monkey pancreata was 1319 islets per gram of pancreatic tissue (range 533-1800) and the usual purity of the preparation varied from 5 to 15 per cent islet tissue. Twelve cynomolgus monkeys underwent total pancreatectomy, preparation of islets from the excised pancreas and autotransplantation to either the spleen or the liver. Three animals received no transplant and became immediately diabetic, surviving 4-8 days. Four animals became normoglycaemic after intrasplenic islet transplantation, and survived 6 weeks, at which time splenectomy was performed with immediate onset of diabetes. Splenic vein insulin sampling confirmed the spleen as the source of insulin, and histological examination showed implanted islet tissue in all cases. Five animals became normoglycaemic after intrahepatic islet implantation. Three animals subsequently became diabetic at 4 and 5 months and two animals still have functioning grafts, the longest function being 9 months. These results suggest that in the cynomolgus monkey sufficient islets can be extracted from a single donor pancreas to reverse diabetes.

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PubMed1986

Long-term normoglycemia in pancreatectomized dogs transplanted with frozen/thawed pancreatic islets.

Storage of pancreatic islets by cryopreservation would greatly facilitate a large scale program of clinical islet transplantation. We report success on long-term follow-up with autotransplantation of frozen/thawed canine pancreatic fragments. Total pancreatectomy and islet isolation by collagenase ductal perfusion and mechanical disruption preceded either acute autotransplantation or cryogenic preservation prior to autotransplantation. Cryopreservation was by dimethylsulfoxide equilibration, cooling at 0.25 degrees C/min to -75 degrees C, storage in liquid N2 and thawing at 3.5 degrees C/min. Four of five acutely autotransplanted dogs remained normoglycemic for 20 months, with three of four maintaining normal K values on intravenous glucose tolerance test (IVGTT) and nondiabetic values on oral GTT. Four of four dogs transplanted with frozen/thawed islets remained normoglycemic for 15 months with three of four maintaining nondiabetic IV GTT K values and normal oral GTTs for 15 months. Both acutely transplanted and frozen/thawed islets are capable of maintaining long-term metabolic control. Cryopreservation preserved viability of sufficient canine pancreatic islets to reverse diabetes with autotransplantation. Function of the frozen-thawed islets showed minimal deterioration during a follow-up of 15 to 18 months.

Animals↗

PubMed1984

Cryopreservation of insulin-producing tissue in rats and dogs.

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Animals↗

PubMed1984

Intrasplenic isografts of canine pancreatic islets: metabolic study.

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Animals↗

PubMed1983

Transplantation of cryopreserved and fresh rat islets and canine pancreatic fragments: comparison of cryopreservation protocols.

(1) The freezing protocol we have devised for rat islets results in normal clinical indices and almost normal glucose tolerance in diabetic recipients of the same inbred strain. (2) Cryopreservation of canine islet-containing pancreatic tissue required a higher temperature than rat islets during exposure to the protective agent. (3) Because of the similar compactness of the pancreas in man and dog, we consider this canine model useful for formulating optimal cryopreservation techniques for the human pancreas. (4) Cryopreservation may partially purify islet-containing tissue of its exocrine content.

Animals↗

PubMed1983

Normoglycemia after reflux of islet-containing pancreatic fragments into the splenic vascular bed in dogs.

Canine pancreata obtained at total pancreatectomy were cannulated via the ducts and perfused with collagenase to prepare a tissue suspension that was isografted into the spleen (preparation congruent to 2 h, mean graft vol = 10 +/- 1 ml containing 24% of the B-cell mass/pancreas). In 13 dogs the tissue was implanted by reflux into terminal splenic veins: two died postoperatively, and in two the intrasplenic vein wall was inadvertently punctured during cannulation. In the remaining nine, mean fasting blood glucose (BG) was less than or equal to 150 mg/dl initially; one was killed at 2 wk (distemper) and one at 6 wk (sepsis, diabetes), and one died at 9 wk (intestinal obstruction). Mean BG was 94 +/- 4 mg/dl at 1 mo and remained in this range until the dogs were killed at 5 mo (91 +/- 13 mg/dl). During glucose-tolerance testing 1 wk preimplantation and 1 mo and 2-3 mo postimplant, mean values were: K (decline in glucose concentration, %/min), 3.4 +/- 0.2, 1.4 +/- 0.1, and 1.5 +/- 0.1; peak insulin (microU/ml), 50 +/- 5, 12 +/- 1, and 11 +/- 2; fasting serum glucagon (pg/ml), 33 +/- 3, 59 +/- 12, and 53 +/- 9, with no change in the glucagon response. Histologically, the spleens contained prominent islets. In five other dogs, the tissue was injected into the splenic pulp: mean BG rose to greater than or equal to 250 mg/dl at 2 wk (compared with initial series, P less than 0.001) and remained elevated until death at 6 wk, when histologic examination of the spleens showed severe fibrosis and no islets. Apancreatic controls (N = 4) survived 10 +/- 3 days; BG was 343 +/- 11 mg/dl terminally. We conclude that this modified method for collagenase perfusion of a single large-mammal pancreas via the ducts provides sufficient viable islets to induce prolonged normoglycemia (5 mo) and preserve the response to glucose challenge. Reflux of pancreatic fragments into splenic veins appears more efficient than intrapulp implantation.

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PubMedDate not supplied

Islet isolation assessment in man and large animals.

Recent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity and in vitro and in vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 mu diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason, in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Animals↗

PubMedDate not supplied

Bacteremia due to transplantation of contaminated cryopreserved pancreatic islets.

OBJECTIVE: To report two cases of pancreatic islet transplantation-related septicemia, and the results of an investigative protocol to identify potential sources of contamination. DESIGN: Case series. SETTING: University hospital clinical investigational islet transplantation program. RESULTS: The last two of our first seven islet transplantation recipients developed Enterobacter cloacae septicemia within hours of islet infusion. Both had received thawed cryopreserved islet infusions. No source of infection apart from islets could be identified. Pancreas harvesting and islet isolation protocols provided multiple opportunities for contamination. Environmental cultures during a mock islet isolation procedure failed to identify a source of Enterobacter. Previously cryopreserved islet lots were thawed and submitted for culture, 14/47 grew micro-organisms including E. cloacae in four instances. Following revision of protocols for aseptic handling of islets during processing and cryopreservation 55 consecutive pancreata undergoing processing were evaluated; 7 grew micro-organisms on arrival and in 3 cases these persisted through to cryopreservation. CONCLUSION: Two of seven islet transplantation recipients developed septicemia, likely related to infusion of contaminated cryopreserved islets. Using existing technology, for isolating islets from donor pancreata, recipients will remain at risk for this complication. Prevention should entail strict adherence to aseptic technique, and, possibly, use of surveillance microbial cultures during the islet isolation process.

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PubMedDate not supplied

Bulk cryopreservation of isolated islets of Langerhans.

Current methods to isolate human islets of Langerhans are limited and multiple donors are required for successful reversal of longstanding Type 1 diabetes mellitus. Cryopreservation of isolated islets is an effective method of storing and pooling islets. Current cryopreservation protocols are cumbersome due to current practices of placing small aliquots of islets per individual freezer tube. In the present study, we examined the application of a blood freezer bag for the cryopreservation of isolated islets by slow cooling and rapid thawing. Freezing and thawing profiles generated using thermocouples placed inside a 500 mL Cryocyte (Baxter) blood freezer bag showed that a longer equilibration period at -7.4 degrees C was necessary to consistently achieve nucleation and cooling profiles similar to those observed in glass tubes. When known numbers of rat islets were placed in the freezer bag and the cryoprotectant dimethyl sulfoxide (DMSO) was added in a stepwise fashion and removed using a sucrose dilution, the islet recovery compared with glass tubes was 92 +/- 4.8 vs. 90 +/- 2.3% (n = 4, p = ns, Mann-Whitney U-test). When purified canine islets were cryopreserved in a single freezer bag or in multiple glass tubes, the recovery was similar (78.8 +/- 12.5% recovery for freezer bag vs. 82.3 +/- 5.3% for glass tubes; n = 6, p = ns). In vitro function was equivalent for both groups. The stimulation index of insulin release during glucose perifusion (stimulated over basal insulin secretion) for canine islets cryopreserved in a freezer bag vs. glass tubes was 3.2 +/- 1.0 and 2.3 +/- 1.3, respectively (n = 6, p = ns). These values were significantly lower than the nonfrozen control islets (6.9 +/- 2.4, p < 0.05). When 2000 canine islets cryopreserved in either a freezer bag, or glass tubes were transplanted into diabetic nude mice, the animals became and remained normoglycemic posttransplant. We conclude that the survival of freshly isolated canine islets cryopreserved in a single freezer bag is equivalent to the glass tube method. Bulk cryopreservation of islets in a single freezer bag will facilitate effective low temperature tissue banking to support ongoing clinical trials of islet transplantation.

Animals↗

PubMedDate not supplied

Development of an automated computer-controlled islet isolation system.

Before clinical islet transplantation can become an effective and reliable treatment for type 1 diabetic patients, there must be significant improvements in the methods employed for the isolation of islets of Langerhans. We have developed an automated cell extraction system (ACES), which allows computer control of the isolation process. As well, it incorporates a novel method of recombining dissociated pancreatic tissue. Following initial system design and testing to determine the optimal system configuration, a series of 12 consecutive canine islet isolations were performed. Pancreases were perfused with collagenase via the duct and dissociated and recombined using either the standard Ricordi-based protocol (group 1, n = 6) or dissociated and recombined using the ACES system (group 2, n = 6). A total of 90.8 +/- 21 x 10(3) islet equivalents (IE) (mean +/- SEM) were recovered in group 1 vs. 99 +/- 14 x 10(3) IE in group 2 (p = NS, student unpaired t-test). Following Ficoll purification the recovery was 56.2 +/- 14 x 10(3) IE for group 1 vs. 54.7 +/- 11 x 10(3) IE for group 2 (p = NS). Viability was equivalent with an 8.6-fold increase in insulin secretion for group 1 and an 8.8-fold increase for group 2 when the islets were exposed to high glucose solution supplemented with IBMX (3-isobutyl-1-methylxanthine) during static incubation. In vivo function was equivalent following transplantation of 2000 IE under the kidney capsule of alloxan-induced diabetic nude mice with five of six and five of seven mice surviving long-term (> 50 days posttransplant) (groups 1 and 2, respectively). This data shows that an entirely automated pancreatic islet extraction system can result in effective canine islet recovery without compromising islet yields and viability. The ACES system has several advantages over the standard isolation protocol. These include: 1) computer control and monitoring over all phases of the isolation, 2) a single-use sterile disposable tubing set, and 3) a novel method of tissue recombination.

Animals↗

PubMedDate not supplied

Comparison of methods used for the removal of DMSO following cryopreservation and the development of an automated protocol.

Current methods of islet isolation are limited, thus requiring islets to be pooled from multiple donors to provide sufficient islet mass to permit insulin independence following islet transplantation. Low temperature banking is one approach used to pool islet preparations. Recently, we developed a method for bulk cryopreservation of islets in a single freezer bag system that is less labor-intensive and more readily kept sterile. As a further improvement to this bulk cryopreservation protocol we examined islet survival following slow-step dilution or our standard sucrose dilution protocol. Known numbers of canine islets were cryopreserved in DMSO by slow cooling to -40 degrees C, storing at -196 degrees C, and rapid thawing. When islets were frozen and thawed in glass tubes the recovery of islets after 48 h of tissue culture was significantly higher when the DMSO was removed using either a slow step (71.7 +/- 2.7%) or a modified slow step (75.7 +/- 3.9%) protocol as compared with the standard sucrose dilution protocol (65.7 +/- 3.0%) (p < 0.05, unpaired t-test). Insulin secretion in vitro and in vivo graft function was similar between the experimental groups. Similarly, when islets were frozen then thawed in freezer bags, islet recovery following 48 h postcryopreservation tissue culture at 37 degrees C was 74.8 +/- 2.4% for slow-step dilution compared with 66.2 +/- 2.7% for the standard sucrose dilution group (p < 0.05, unpaired t-test). Islets thawed in the freezer bag using the modified slow-step dilution protocol showed equivalent functional viability during static incubation to nonfrozen controls. Bulk cryopreservation of isolated islets in single blood freezer bags is a practical alternative to cryopreservation in glass tubes. Development of an automated protocol for the slow stepwise removal of the cryoprotectant from islets in freezer bags will facilitate low temperature tissue banking of islets.

Animals↗

PubMedDate not supplied

Effect of cryopreservation on the survival and function of murine islet isografts and allografts.

We compared the efficacy of fresh and frozen/thawed islets by determining the minimum number required to consistently reverse diabetes in mice. Defined numbers of islets, isolated from Balb/c (H-2d) and CBA/J (H-2k) mice, were transplanted into streptozotocin-induced diabetic Balb/c mice. Frozen/thawed grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. At 100 days after transplantation, isografts were recovered for measurement of insulin content. Mean (+/-SD) recovery of cryopreserved islets after thawing was 80 +/- 3% (range 67-89%). For both fresh and frozen/thawed isografts and allografts, 200 islets were required to establish normoglycemia. The degree of metabolic function provided by equivalent quantities of fresh and frozen/thawed grafts was similar; and all normoglycemic isograft recipients remained so until graft nephrectomy. The insulin content of fresh and frozen/thawed isografts containing 200 and 300 islets were 151 +/- 25 and 126 +/- 8 mU and 259 +/- 36 and 278 +/- 20 mU, respectively. Among allograft recipients, median survival ranged from 15 to 20 days, and was not influenced by cryopreservation or graft size. The results of this study demonstrate a high rate of recovery of viable islets following cryopreservation. The function of equivalent quantities of fresh and cryopreserved islet isografts and allografts in nonimmunosuppressed recipients is similar.

Animals↗