Efficacy Comparison Between Intraportal and Subcapsular Islet Transplants in a Murine Diabetic Model

BackgroundIt is important to determine the efficacy of intraportal (IP) islet transplantation in comparison with other transplant sites. In this study, we sought to determine the optimal number of islets to achieve normoglycemia following transplantation into the liver versus the kidney using a mouse model.MethodsStreptozotocin-induced diabetic mice (Balb/C) were transplanted with syngeneic islets via the IP versus renal subcapsular (SC) routes. The transplanted islet numbers were 0 to 800 (n = 3–5). We assessed the correlation between parameters and islet numbers, comparing IP versus SC groups. The parameters were: (1) percentage of normoglycemia; (2) postoperative days to normoglycemia; (3) mean blood glucose levels at various points from pretransplantation to the end of the study (postoperative day 28); (4) mean serum insulin; and (5) area under the curve of blood glucose levels after glucose injection.ResultsTwo hundred islets yielded normoglycemia in renal subcapsular grafts, while 800 islets were the minimum required for normoglycemia with IP transplantation. The transplant efficacy in SC transplantation was 2 to 5 times greater than that of IP transplantation. The days to normoglycemia were significantly different between IP versus renal SC islets (13.25 ± 4.38 days vs 4.50 ± 0.81 days; P = .007).ConclusionThe efficacy of islet transplantation in murine diabetic models was significantly greater under the kidney capsule. Clinical islet transplantation could benefit from trials of alternative transplant sites.     

Evaluation of a Purified Enzyme Blend for the Recovery and Function of Canine Pancreatic Islets

Recently developed technologies enabling the production of a reproducible, purified enzyme blend for optimal human pancreatic islet isolation has renewed interest in clinical islet transplantation. The canine model has been an ideal preclinical model for the development of islet transplantation protocols. As seen in other species, the application of crude collagenase for isolating canine islets resulted in highly variable islet yields, extensive islet fragmentation, and variable islet functionality. We compared the function of commercially available crude collagenases with that of Liberase™-CI purified enzyme blend for canine islet isolation. We also compared two manufacturing runs of Liberase-CI enzyme (lots 1 and 2) to demonstrate reproducibility of islet recovery and function. We report on the improved recovery and function of islets isolated using Liberase-CI enzyme. No difference in dog age, mean body weight, or pancreas weight were observed between the experimental groups. We observed a significantly higher postpurification recovery of islet equivalent number (IE) from pancreases processed using two lots of Liberase-CI enzyme (189 ± 20 × 103 IE, n = 4) and lot 2 (234 ± 39 × 10³ IE, n = 7) than that obtained from pancreases processed with Sigma Type V (116.8 ± 27 × 10³ IE, n = 5), Serva collagenase (49 ± 11.6 × 10³ IE, n = 5, p < 0.05) or Boehringer–Mannheim (BM) Type P collagenase (85.4 ± 25 × 10³ IE, n = 5, p < 0.05, ANOVA). No significant differences were observed in islet yield recovery from pancreases processed using the two production lots of Liberase-CI enzyme. Islet survival after 48 h in culture at 37°C was significantly higher from islets isolated using Liberase-CI enzyme (88 ± 3.7% survival) when compared to Sigma Type V (81.8 ± 3.3%), Serva (71.7 ± 2.8%), and BM Type P (77 ± 7.2%) (p < 0.05). Islet functional testing in vitro demonstrated islets isolated using crude collagenase had an increased insulin basal release and a reduced insulin stimulated response when compared with islets isolated using the two lots of Liberase-CI enzyme. The calculated stimulation index was 7.8 ± 1.7, 3.1 ± 0.6, and 4.8 ± 1.1 for Sigma Type V, Serva, and BM Type P isolated islets, respectively, compared to 15.7 ± 1.6 and 16.2 ± 1.9 for islets isolated with Liberase-CI enzyme production lots 1 and 2, respectively (p < 0.05). This evaluation demonstrates that a purified enzyme blend can significantly improve islet recovery and function. It also demonstrates the manufacturing reproducibility of Liberase-CI enzyme lots resulting in the isolation of canine islets with the same degree of efficacy. A blend of purified enzymes, specifically formulated for canine islet isolation, can consistently yield large numbers of islets that survive longer in culture and demonstrate an improved insulin response in vitro.     

Effect of Etanercept Concentration on Human Islet Integrity

Background

Etanercept is widely used as an antiinflammatory drug to improve engraftment after intraportal islet transplantation. In contrast to other immunosuppressive agents, very little is known about detrimental effects of etanercept on islets. The aim of this pilot study was to define the toxic range of etanercept.

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°C, storing at -196°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°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 step-wise removal of the cryoprotectant from islets in freezer bags will facilitate low temperature tissue banking of islets.     

A prospective comparison of discontinuous EuroFicoll and EuroDextran gradients for islet purification

Density gradient separation of islets from exocrine tissue is usually performed with Ficoll. However, this reagent adds significantly to the cost of the isolation. The aim of this study was to evaluate the performance of Dextran as a potential low-cost substitute for Ficoll and to evaluate the effects of cold storage of the pancreatic digest prior to purification. Pancreases were procured from mongrel dogs, loaded with collagenase and mechanically dissociated. Washed pancreatic digest was collected and divided into two fractions that were purified using discontinuous gradients on the Cobe 2991 processor using identically prepared EuroFicoll (EF) or EuroDextran (ED) gradients. Alternate groups were suspended in EC and stored on ice, while the other fraction were resuspended in the 1.108-g/mL gradient layer (either EF or ED) and loaded into the COBE. This tissue layer was overlaid with layers of densities 1.096 and 1.037 g/mL and a HBSS cap, and centrifuged for 5 min at 800 × g. Purified islets were collected from the interface between the 1.037 and 1.096 layers and islet recovery, purity, and function were assessed. From a series of eight isolations, 72.9 ± 8.2% (mean ± SEM) of the islets were recovered from the EF purified gradients compared with 62.6 ± 8.3% from ED gradients (p = NS, paired t-test). Gradients of ED that were run following hypothermic storage of the digest in cold EC solution (stored ED) had reduced islet recovery when compared with islet recovery from gradients prepared in EF(stored EF) (51.6 ± 9.6% for ED stored vs. 72.9 ± 11.9 for EF stored, p < 0.05). Islet recovery from EF gradients was equivalent between the stored and nonstored groups. The purity of preparations from the stored ED gradients was also reduced (71.3 ± 4.3%) when compared with islets that were immediately purified after dissociation (82.5 ± 4.8%, p < 0.05). Static glucose stimulation assay showed equivalence between the islets from ED and EF gradients. The stimulation index (SI) was 9.3 ± 0.9 for EF islets compared with 7.9 ± 1.4 for ED islets for digest purified immediately. However, if the digest was hypothermically stored in EC solution, a decrease in functional viability was observed in both the EF and the ED groups (7.7 ± 1.4 and 5.9 ± 0.8, respectively). Out of five alloxan-induced diabetic nude mice transplanted under the kidney capsule with 2000 islets isolated from the nonstored groups, three remained euglycemic >50 days posttransplant with either EF or ED islets. These experiments demonstrate effective recovery of equivalent numbers of canine islets using discontinuous gradients of ED or EF immediately following enzymatic digestion. However, following storage of the digest in cold EC solution results in a reduction in both islet recovery and function when gradients of ED are utilized.     

Gastric submucosal alleviated pro-inflammation cytokines mediated initial dysfunction of islets allografts

BackgroundThe liver and renal capsule are the most common site for experimental pancreatic islet transplantation, but it is not optimal. Gastric submucosa space may be an ideal site for islet transplantation; however, whether pro-inflammation factors mediated islet dysfunction could be avoided or alleviated is still unclear.MethodsIslets of Sprague Dawley (SD) rat were transplanted into the streptozotocin-induced diabetic SD rats. Transplantation sites included gastric submucosa (GS), intraportal vein (PV) and kidney capsule (KC), and the efficiency of glycemic control and site-specific differences of islet grafts were compared.ResultsWith limited number of islets (800 IEQ) transplanted, improvement of recipient glycometabolism was superior in the GS group. When transplanted with 1200 IEQ islets, the survival of islet grafts were significantly prolonged in the GS group (25.87 ± 4.08 days, compared to 15.97 ± 0.83 days and 17.33 ± 1.41 days in PV and KC groups, respectively, P < .05). Compared with the PV group, the levels of IL-1β and TNF-α were significantly depressed in GS group after 12 h transplantation (15.5 ± 0.70 pg/mL and 13.28 ± 2.80 pg/mL vs. 262.26 ± 53.37 pg/mL and 138.51 ± 39.58 pg/mL, P < .05).ConclusionsGastric submucosal would be a potential ideal site for islet transplantation in rat. Gastric submucosal might alleviate the early islet dysfunction triggered by the IL-1β and TNF-α, and which requires a low number of transplanted islets and have a good glycemic control in return.     

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.     

Co-transplantation of Xenogeneic Bone Marrow–derived Mesenchymal Stem Cells Alleviates Rejection of Pancreatic Islets in Non-obese Diabetic Mice

Bone marrow–mesenchymal stem cells (BM-MSCs) have generated a great perspective in the field of regenerative medicine, and also in the treatment of inflammatory and autoimmune diseases in the past decade due to their immunomodulatory and anti-inflammatory properties. Here, we investigated the effect of xenogeneic BM-MSCs and pancreatic islets co-transplantation obtained from Wistar rats in preventing rejection or inducing tolerance to islet transplantation in non-obese diabetic mice. Non-obese diabetic mice were treated with co-transplantation of pancreatic islets and BM-MSCs (islet + MSCs group) or pancreatic islets only (islet group). Compared to the islet group, islet + MSCs had a lower expression of inflammatory markers, such as, tumor necrosis factor– α (13.40 ± 0.57 vs. 9.90 ± 0.12, P = .01), monocyte chemoattractant protein 1 (51.30 ± 6.80 vs. 9.00 ± 1.80, P = .01), and interleukin 1β (IL-1β) (16.2 ± 1.65 vs. 6.80 ± 1.00, P = .04). Comparing the expression of immune tolerance markers, it is noted that animals receiving the co-transplantation showed a significantly higher expression than the islet group of IL-4 (25.60 ± 1.96 vs. 2.80 ± 0.20, P = .004), IL-10 (188.40 ± 4.60 vs. 4.55 ± 0.12, P = .0001), and forkhead box P3 (34.20 ± 1.3 vs. 1.30 ± 0.2, P = .004), respectively. These results suggest an immunomodulatory action of BM-MSC in islet xenotransplantation showing that these stem cells have the potential to mitigate the early losses of grafts, due to the regulation of the inflammatory process of transplantation.     

Hypogastrinemia in streptozotocin diabetes with islet transplantation-reconstitution

Gastrin is present in normal mammalian pancreatic islets, as well as in the antrum and duodenum. Serum gastrin levels are responsive to many physiologic and pharmacologic factors including hyperglycemia, somatostatin, and glucagon. To evaluate the effects of streptozotocin diabetes and islet transplantation on gastrin homeostasis, young, adult, male Lewis rats underwent streptozotocin diabetes alone (N = 14), diabetes plus intraperitoneal islet isografts (N = 22), or sham operation alone (N = 18). Streptozotocin reduced fasting gastrin immunoreactivity (107 pg/ml ± 26 mean ± SEM) compared to controls (256 pg/ml ± 31) (P < 0.001). Islet isotransplantation resulted in restoration of fasting gastrin immunoreactivity (230 pg/ml ± 19) to levels significantly greater than diabetics (P < 0.001) and comparable to control animals (P = NS). Normalization of serum gastrin levels occurred within one month of transplantation and persisted for up to 14 months. In addition, media from cultures of $\text{4}\text{7}$ dispersed neonatal rat pancreas cultures contained gastrin immunoreactivity. Streptozotocin diabetes produces hypogastrinemia; intraperitoneal islet isotransplantation normalizes fasting gastrin immunoreactivity. Rat islet culture medium contains gastrin-like immunoreactivity. Pancreatic islets would appear to produce or control a portion of circulating gastrin immunoreactivity.     

Pancreatic islet isolation after gastric bypass in a rat model: technique and initial results for a promising research tool

BackgroundRoux-en-Y gastric bypass (RYGB) affords a high remission rate of type 2 diabetes mellitus among morbidly obese diabetic patients. We report the use of the isolated islet technique to assess pancreatic function and glucoregulatory mechanisms after RYGB surgery.MethodsA total of 15 adult, male, Sprague Dawley diet-induced obese rats were randomly divided into 3 experimental groups: sham, RYGB, and pair-fed, with 5 rats in each group. The body weight was measured at baseline and every week for 4 weeks. Pancreatic islet function was assessed in vitro according to the amount of insulin secreted from isolated islets incubated in 2 mM and 20 mM glucose for 1 hour at 37°C. Fasting plasma glucose, insulin, glucagon-like peptide-1, PYY3-36, and glucose-dependent insulinotropic peptide were measured at baseline and 28 days after surgery.ResultsThe baseline body weight was 917 ± 61, 831 ± 42, and 927 ± 43 g for the sham, RYGB, and pair-fed groups, respectively. The RYGB group lost 32% body weight compared with 16% for the sham and 24% for the pair-fed groups. Glucose-stimulated insulin secretion from the isolated islets in the RYGB group was greater than in the comparison groups (P = .04) at 4 weeks after surgery. Fasting plasma glucagon-like peptide-1 and PYY3-36 were significantly increased at 4 weeks in the RYGB group.ConclusionIslet isolation and stimulation in the present animal model was feasible, affords a direct measurement of pancreatic islet function, and might provide a useful tool to study the effects of RYGB on pancreatic function and the relationship between islet cell function and incretin production after bariatric surgery.     

The sequential combination of a JNK inhibitor and simvastatin protects porcine islets from peritransplant apoptosis and inflammation

Intraductal administration of a c-Jun NH(2)-terminal kinase (JNK) inhibitor enhances islet viability. However, its role in reducing the inflammatory response in islets is unknown. It is also unknown whether a JNK inhibitor could act in synergy with statins. We examined if the sequential combination of a JNK inhibitor and simvastatin would reduce islet inflammation and improve islet viability. We performed porcine islet isolation with or without intraductal administration of SP600125, a JNK inhibitor. This was followed by culture medium supplementation with either nicotinamide alone or nicotinamide plus simvastatin. We assessed the viability of islets by flow cytometry, islet loss during overnight culture, graft function in NOD/SCID mice, and expression of inflammation-related genes in islets. The sequential combination of a JNK inhibitor and simvastatin increased the β-cell viability index of porcine islets cultured overnight (p = 0.015) as well as islet viability as assessed by a DNA binding dye staining (p = 0.011). The combination of a JNK inhibitor and simvastatin significantly increased the islet survival rate (p = 0.027) when the histomorphometry of donor pancreas indicated a large islet proportion of greater than 50.55%. When we transplanted the same islet mass per recipient for each group, there was no difference in overall islet graft function. Intraductal administration of JNK inhibitor significantly suppressed mRNA expression levels of interleukin-1β (IL-1β), interferon-γ, tumor necrosis factor-α, IL-6, IL-8, and macrophage chemoattractant protein-1. It also decreased the concentration of IL-1β (p = 0.040) and IL-8 (p = 0.023) in the culture supernatant. In conclusion, the sequential combination of a JNK inhibitor and simvastatin protected porcine islets from peritransplant apoptosis. Inhibition of JNK reduced the inflammatory response and could be considered an alternative target for suppression of porcine islet inflammation.     

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°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 = us, 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.     

Adenovirus-Mediated Gene Expression of the Human c-FLIPL Gene Protects Pig Islets Against Human CD8+ Cytotoxic T Lymphocyte-Mediated Cytotoxicity

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP_S/L genes, which are potent inhibitors of death receptor–mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP_L seem to be significantly stronger than those of c-FLIP_S. Accordingly, in the present study, expression of c-FLIP_L was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% ± 15.9% and 64.0% ± 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP_L gene were markedly protected from lysis (30.5% ± 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP_L with adenovirus. Pig islets expressing c-FLIP_L are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.     

Improved Islet Survival and Funtion With Rat Endothelial Cells In Vitro Co-Culture

Objective

Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To preserve its function, transplanted islets must be revascularized because arterial and venous connections are disrupted during islet isolation. The current paradigm is that islet revascularization originates from the transplant recipient. This study was designed to test whether the function of isolated islets can be retained by co-culture with thoracic aorta endothelial cells in vitro.

Methods

Sprague-Dawley rats were used in this study. The endothelial cells (ECs) were isolated from the thoracic aorta. The viability of the isolated islets was assessed by acridine orange/propidium iodide (AO/PI) double staining. The islets were either placed in standard cultures (group A) or in co-cultures with ECs (group B). Islet viablity was assessed by an insulin release assay.

Results

The islets in group B exhibited normal morphology with >90% staining positive as detected by AO/PI with 7 days. Insulin release assays showed a significantly higher simulation index (SI) in group B compared with group A (P < .05) except on the first day.

Conclusion

This study suggested that co-cultrue of freshly isolated rat islets with ECs improves postculture survival and islet function in vitro.     

Development of an automated computer-controlled isletisolation 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 × 10³ islet equivalents (IE) (mean ± SEM) were recovered in group 1 vs. 99 ± 14 × 10³ IE in group 2 (p = NS, student unpaired t-test). Following Ficoll purification the recovery was 56.2 ± 14 × 10³ IE for group 1 vs. 54.7 ± 11 × 10³ 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-l-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 advan tages 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.     

Pretreatment of Donor Pigs With a Diet Rich in Soybean Oil Increases the Yield of Isolated Islets

Introduction

The pig is considered the donor species of choice for islet xenotransplantation. However, isolation of porcine islets is difficult, particularly from young pigs. Early life exposure to a high-fat diet (HFD) reportedly encourages islet β-cell expansion in neonatal rodents and improves islet viability in culture from pretreated weanling pigs. In this study, we examined the influence of young donor pretreatment with a soybean oil–enriched HFD on porcine islet mass and yield after islet isolation.

Materials and Methods

Postweaning and between days 70 and 250, pigs were fed either a standard diet (control group; n = 5) or an HFD (experimental group; n = 6). Biochemical blood parameters and acute C-peptide response to intravenous glucose were monitored before pancreas procurement. The study was blinded to objectively evaluate the influence of treated diet. After procurement, pancreas biopsy samples were taken from control and pretreated donor pigs to assess islet number by using a dithizone scoring method and histologic islet area fraction determination. Control and HFD donor pig islets were isolated by using our standard isolation protocol to determine islet yield. Islet isolation characteristics and islet quality were assessed in both groups, and the results were compared.

Results

There were no significant differences in the donor characteristics (age, body weight, glucose disposal rate, acute C-peptide response to intravenous glucose, cholesterol, and aspartate aminotransferase) except fasting blood glucose level between the control and treatment groups (84 ± 6 vs 99 ± 12 mg/dL; P = .0317). The stimulated insulin and C-peptide levels between groups were similar. However, the dithizone score was slightly higher in the treatment group compared with the control group (95.4 ± 38.5 vs 62.6 ± 23.9; P = .1208). Digestion time, digested pancreas weight, pellet volume, and the fragility index were similar in both groups. However, the average islet count (islet equivalent number/g pancreas) at the digest level was significantly higher in the HFD group than in the control group (1578 ± 994 vs 738 ± 202; P = .0344). The functional viability of 2- and 7 day-cultured islets, as assessed by using oxygen consumption rate corrected for DNA, was similar in both groups.

Conclusions

Pretreatment of pigs with HFD enriched with soybean oil could potentially be used to improve the islet mass in donor pigs. Further studies are needed to confirm and optimize the use of HFD for the purpose of increasing islet yield from young donor pigs.     

Ginsenoside Rg3 Enhances Islet Cell Function and Attenuates Apoptosis in Mouse Islets

Background

The transplantation of isolated islets is thought to be an attractive approach for curative treatment of diabetes mellitus. Panax ginseng has been used in oriental countries for its pharmacologic effects, such as antidiabetic and antiinflammatory activities. 20(S)-ginsenoside Rg3 (Rg3), an active ingredient of ginseng saponins, has been reported to enhance insulin secretion–stimulating and antiapoptotic activities in pancreatic beta cells. We performed this study to examine the hypothesis that preoperative Rg3 administration can enhance islet cell function and antiapoptosis before islet transplantation.

Methods

Balb/c mice were randomly divided into 2 groups according to the administration of Rg3 after islet isolation. Mouse islets were cultured in medium supplemented with or without Rg3. In vitro, islet viability and function were assessed. After treatment of islets with a cytokine cocktail (tumor necrosis factor α, interferon-γ, and interleukin-1β), cell viability, function, and apoptosis were assessed.

Results

Cell viability was similar between the 2 groups. Islets cultured in medium supplemented with Rg3 showed 2.3-fold higher glucose-induced insulin secretion than islets cultured in medium without Rg3. After treatment with a cytokine cocktail, glucose-induced insulin release, total insulin content of islets, and apoptosis were significantly improved in Rg3-treated islets compared with cytokine-treated islets. Cytokine-treated islets produced significantly higher levels of nitric oxide (NO) than islets treated with Rg3.

Conclusions

These results suggest that preoperative Rg3 administration enhanced islet function before islet transplantation and attenuated both cytokine-induced damage associated with NO production and apoptosis. Rg3 administration might be a prospective management to enhanced islet function and ameliorate early inflammation after transplantation.     

Repeated intraportal injections of subtherapeutic islet cell isografts restore normoglycemia in streptozotocin-diabetic rats

Poor engraftment and consequent loss of β-cell mass could be one of the factors that are responsible for function loss after intraportal islet transplantation (Tx). Streptozotocin-diabetic rats were transplanted with syngeneic islets, which were injected into the portal vein via an indwelling catheter connected to a subcutaneous port. In Group I (n = 6), 1,000 islets were injected in a single dose into the liver. In Group II (n = 6), five doses of 200 islets were repeatedly injected over a period of 14 days, for a total of 1,000 islets. In Group III (n = 4), five decreasing doses of islets were injected over a period of 14 days, for a total of 750 islets. Nonfasting blood glucose (n-FBG) and body weight (b.wt.) were determined twice a week and an intravenous glucose tolerance test (IVGTT) was performed at 30 and 90 days. In Group I, n-FBG decreased in 2 wk from the time of first islet injection, averaging 110 ± 21.9 mg/dL at 1 mo (p < 0.05 vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group II, n-FBG was normalized in 2 wk averaging 90.2 ± 25 mg/dL on day 12 (p = NS vs. normal controls) and 75.8 ± 14.6 mg/dL at 1 month (p = NS vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group III, n-FBG decreased to normal values in 2 wk, averaging 77 ± 15.7 mg/dL at 1 mo (p = NS vs. normal controls), but normoglycemia was maintained for 40 days and then followed by a progressive increase. Only in Group II, K_G (percent/min decline in glucose level) was not significantly different from that of normal controls (1.702 ± 0.531 at 1 mo and 1.676 ± 0.891 at 3 mo), while it was significantly lower than normal controls in both Group I and III animals. Body weight increase after Tx correlated with the number of transplanted islets and at 90 days, Group III rats showed less increase than Groups I and II (p < 0.05), while no significant differences in b.wt. were recorded between Group I and II. The findings indicate that intraportal islet Tx, injected repeatedly and in small doses, produced better metabolic effects than injection of the same total number of islets in a single dose.     

High yield of rodent islets with intraductal collagenase and stationary digestion—A comparison with standard technique

Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observer ‘blinded’ to the method of isolation used, and only intact islets of diameter ≥ 100 μm were included. Stationary gave 719 ± 114 islets per pancreas (mean ± SD, n = 21 isolations) vs. 487.5 ± 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (≤ 8 mmol/L) by 3.3 ± 4.8 days vs. 1.6 ± 1.5 days for Chopped recipients (p = 0.4 ns, mean ± SEM). IVGTT performed at 1 mo posttransplant gave Kvalues for Stationary of 2.64 ± 0.8 vs. 2.62 ± 0.8 for Chopped (mean ± SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 ± 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.     

Pretreatment of Crude Pancreatic Islets With Mitomycin C (MMC) Prolongs Islet Graft Survival in a Xenogeneic Rat-to-Mouse Model

In this study, we examined the effect of mitomycin C (MMC) treatment on graft survival and evaluated its efficacy in immunomodulation of islet graft for transplantation. Male WS rats were used as islet donors and streptozotocin-induced diabetic C57BL/6 mice as recipients. The isolated islets were treated with MMC at concentrations of 0, 0.1, 1, 3.2, 10, 32, 100, 320, and 1000 μg/mL for 30 min, and were cultured for 20 h. Then, 300–400 islets were transplanted into the renal subcapsular space of diabetic mice. Significant prolongation of graft survival was obtained when the islets were treated with MMC at a concentration of 10, 32, or 100 μg/mL (MST 23 ± 7.4, 17.5 ± 5.4, 29.6 ± 9.7 days: p < 0.003, p < 0.012, p < 0.001, respectively, vs. 12.3 ± 2.7 days for culturing alone). Islets treated with MMC at a concentration of 320 μg/mL or more failed to restore normoglycemia in the diabetic recipient mice after transplantation. Viability of islets incubated with doses up to 100 μg/mL, assessed under the confocal microscope after propidium iodide and Hoechst 33342 staining, was maintained well comparable to that of freshly isolated islets, while those treated at 320 μg/mL was significantly decreased. Thus, a therapeutic window for MMC efficacy was found at concentrations from 10 μg/mL to 100 μg/mL. This modality is simple and effective and underlying molecular mechanisms need to be determined in the future.