Search

We previously reported that modified extracellular-type trehalose-containing Kyoto (MK) solution, which contains a trypsin inhibitor (ulinastatin), significantly improved the islet yield compared with University of Wisconsin (UW) preservation, which is the gold standard for organ preservation for islet isolation. In this study, we evaluated the efficiency of a modified histidine-lactobionate (MHL) solution in addition to UW or MK solution. The MHL solution has a high sodium-low potassium composition with low viscosity compared with the UW solution. Moreover, similar to MK solution, MHL solution also contains ulinastatin.Porcine pancreata were preserved in UW, MK, or MHL solution, followed by islet isolation. An optimized number (1500 IE) of isolated islets from each group were then transplanted into streptozotocin-induced diabetic mice.The islet yield before and after purification was significantly higher in the MHL group than in the UW group. On the contrary, the islet yield before and after purification was not significantly different between the MHL and MK groups. Preserving the porcine pancreata in MHL solution improved the outcome of islet transplantation in streptozotocin-induced diabetic mice compared with that in UW solution.Pancreas preservation with MHL solution preserves islet function better than UW solution. The effect of MHL solution is similar to that of MK solution, suggesting that MHL solution can be used as an alternative to MK solution for pancreatic islet transplantation.

BACKGROUND Although the liver is the primary site for clinical islet transplantation, it poses several restrictions, especially limited tissue volume due to portal vein pressure. We evaluated the preperitoneal space as an extrahepatic islet transplant site to deliver high tissue volumes and sustain long-term graft function. METHODS A peritoneal pouch was formed by dissecting the parietal peritoneum from the transversalis fascia of mice. Syngeneic C57BL/6 donor islets were transplanted into the peritoneal pouch of diabetic mouse recipients. Blood glucose was monitored for islet function, and miR-375 was analyzed for islet damage. Islet graft morphology and vascularization were evaluated by immunohistochemistry. [F] fluoro-D-glucose positron emission tomography/computed tomography was used to image islet grafts. RESULTS Transplantation of 300 syngeneic islets into the peritoneal pouch of recipients reversed hyperglycemia for >60 days. Serum miR-375 was significantly lower in the peritoneal pouch group than in the peritoneal cavity group. Peritoneal pouch islet grafts showed high neovascularization and sustained insulin and glucagon expression up to 80 days posttransplantation. A peritoneal pouch graft with high tissue volume (1000 islets) could be visualized by positron emission tomography/computed tomography imaging. Human islets transplanted into the peritoneal pouch of diabetic nude mice also reversed hyperglycemia successfully. CONCLUSIONS Islets transplanted into a dissected peritoneal pouch show high efficiency to reverse diabetes and sustain islet graft function. The preperitoneal site has the advantages of capacity for high tissue volume, enriched revascularization and minimal inflammatory damage. It can also serve as an extrahepatic site for transplanting large volume of islets necessitated in islet autotransplantation.

Background Islet transplantation is a promising treatment in patients with complicated diabetes. The ideal transplant site that can extend islet graft survival and reduce the required number of engrafted islets remains to be established. Methods Donor islets were isolated from red fluorescent protein (RFP) mice and transplanted into interscapular brown adipose tissue (BAT) or unilateral inguinal white adipose tissue of age-matched diabetic RFP mice. Blood glucose and body weight of the mice were monitored, and vitality and function of ectopic RFP islets were detected by fluorescence imaging, histological examination, and intraperitoneal glucose tolerance test (GTT). Results BAT enabled the marginal number of grafted islets (80 islets) to restore blood glucose, insulin level, and GTT to normal values in all diabetic recipient mice in the short term after graft, and maintained these values for 1 year at the end of the experiment. Importantly, in the short term after transplantation, abundant extra- and intraislet neovasculatures were observed in BAT, but not in white adipose tissue, which allowed the ectopic islets to retain typical architecture and morphology and contributed to the normal GTT. Moreover, the islet-engrafted BAT displayed normal structure and morphology without significant immunocyte infiltration, and the recipient mice also showed normal lipid levels in the blood. Conclusions BAT remarkably enhances the viability and biological function of the transplanted ectopic islets. Moreover, the anatomical location of BAT lends itself to biopsy, removal, and islet retransplantation, which strongly suggests the BAT as a potential desirable site for islet transplantation in basic and clinical research.

Background Transplantation of pancreatic islets into subcutaneous cavities in diabetic rats may be as, or even more effective than transplantation into the portal vein. Identifying the optimal timing of the individual steps in this procedure is critical. Methods Macroporous scaffolds were placed in the subcutaneous tissue of diabetic male Lewis rats for 7 or 28 days and the healing of the tissue inside the scaffolds was monitored. A marginal syngeneic graft comprising 4 islets/g of recipient body weight was transplanted in the best timing focusing mainly on vascularization. Recipients were monitored for blood glucose levels and tolerance tests. Histological examination was performed in all implanted scaffolds. The presence of individual endocrine cells was analyzed in detail. Results Blood glucose levels remained within the physiological range in all recipients until the end of experiment as well as body weight increase. Coefficients of glucose assimilation were normal or slightly reduced with no statistically significant differences between the groups 40 and 80 days after transplantation. Histological analysis revealed round viable islets in the liver similar to those in pancreas, but alpha cells practically disappeared, whereas islets in the scaffolds formed clusters of cells surrounded by rich vascular network and the alpha cells remained partially preserved. Conclusions Subcutaneous transplantation of pancreatic islets is considerably less invasive but comparably efficient as commonly used islet transplantation into the portal vein. In consideration of alpha and beta cell ratio, the artificial subcutaneous cavities represent a promising site for future islet transplantation therapy.

Background White adipose tissue (WAT) is a candidate transplantation site for islets. However, the mechanism of islet engraftment in WAT has not been fully investigated. In this study, we attempted to clarify the therapeutic effect and mechanism of islet transplantation into visceral WAT. Methods Two hundred mouse islets were transplanted into epididymal WAT of syngeneic diabetic mice by wrapping islets with the tissue (fat-covered group). Mice that received intraperitoneal and renal subcapsular islet transplantations were used as negative and positive control groups, respectively. Results The transplant efficacy, including improvements in blood glucose and plasma insulin levels and in glucose tolerance tests, of the fat-covered group was superior to the negative control group and almost equal to the positive control group. Vessel density of engrafted islets in the fat-covered group was higher than that in the positive control group. It was speculated that the mechanism of islet engraftment in WAT might consist of trapping islets in WAT, adhesion of islets via a combination of adhesion factors (fibronectin and integrin β1), and promotion of angiogenesis in islets by expression of angiogenic factors induced by adiponectin. Conclusions Visceral WAT is an important candidate for islet transplantation. Adhesion factors and adiponectin might contribute to islet engraftment into WAT. Further studies to elucidate the detailed mechanism are necessary.

Background: Islet transplantation is a promising treatment for type-1 diabetes; however, donor shortage is a concern. Even when a pancreas is available, low islet yield limits the success of transplantation. Islet culture enables pooling of multiple low-yield isolations into an effective islet mass, but isolated islets rapidly deteriorate under conventional culture conditions. Oxygen depletion in the islet core, which leads to central necrosis and volume loss, is one of the major reasons for this deterioration. Methods: To promote long-term culture of human islets in PIM-R medium (used for islet research), we adjusted temperature (12, 22, and 37°C) and oxygen concentration (21% and 50%). We simulated the oxygen distribution in islets based on islet oxygen consumption rate and dissolved oxygen in the medium. We determined the optimal conditions for oxygen distribution and volume maintenance in a 2-week culture and assessed viability and insulin secretion compared to noncultured islets. In vivo islet engraftment was assessed by transplantation into diabetic NOD-SCID mouse kidneys. We validated our results using CMRL 1066 medium (used for clinical islet transplantation). Results: Simulation revealed that 12°C–50% oxygen PIM-R culture supplied oxygen effectively into the islet core. This condition maintained islet volume at >90% for 2 weeks. There were no significant differences in viability and function in vitro or diabetic reversal rate in vivo between 2-week cultured and noncultured islets. Similar results were obtained using CMRL 1066. Conclusion: By optimizing temperature and oxygen concentration, we cultured human islets for 2 weeks with minimal loss of volume and function.

During intraportal pancreatic islet transplantation (PITx), early inflammatory reactions cause an immediate loss of more than half of the transplanted graft and potentiate subsequent allograft rejection. Previous findings suggest that cibinetide, a selective innate repair receptor agonist, exerts islet protective and antiinflammatory properties and improved transplant efficacy in syngeneic mouse PITx model. In a stepwise approach toward a clinical application, we have here investigated the short- and long-term effects of cibinetide in an allogeneic mouse PITx model.Streptozotocin-induced diabetic C57BL/6N (H-2) mice were transplanted with 320 (marginal) or 450 (standard) islets from BALB/c (H-2) mice via the portal vein. Recipients were treated perioperative and thereafter daily during 14 d with cibinetide (120 µg/kg), with or without tacrolimus injection (0.4 mg/kg/d) during days 4-14 after transplantation. Graft function was assessed using nonfasting glucose measurements. Relative gene expressions of proinflammatory cytokines and proinsulin of the graft-bearing liver were assessed by quantitative polymerase chain reaction. Cibinetide's effects on dendritic cell maturation were investigated in vitro.Cibinetide ameliorated the local inflammatory responses in the liver and improved glycemic control immediately after allogeneic PITx and significantly delayed the onset of allograft loss. Combination treatment with cibinetide and low-dose tacrolimus significantly improved long-term graft survival following allogeneic PITx. In vitro experiments indicated that cibinetide lowered bone-marrow-derived-immature-dendritic cell maturation and subsequently reduced allogeneic T-cell response.Cibinetide reduced the initial transplantation-related severe inflammation and delayed the subsequent alloreactivity. Cibinetide, in combination with low-dose tacrolimus, could significantly improve long-term graft survival in allogeneic PITx.

Background It has been proposed that islet transplants comprised primarily of small rather than large islets may provide better graft function, due to their lower susceptibility to hypoxic damage. Our aim was to determine whether islet size correlated with in vivo graft function in islet transplant recipients with C peptide–negative type 1 diabetes when islets have undergone pretransplant islet culture. Methods Human pancreatic islets were isolated, cultured for 24 hours and infused by standardized protocols. Ninety-minute stimulated C-peptide concentrations were determined during a standard meal tolerance test 3 months posttransplant. The islet isolation index (IEq/islet number) was determined immediately after isolation and again before transplantation (after tissue culture). This was correlated with patient insulin requirement or stimulated C-peptide. Results Changes in insulin requirement did not significantly correlate with islet isolation index. Stimulated C-peptide correlated weakly with IEq at isolation (P = 0.40) and significantly with IEq at transplantation (P = 0.018). Stimulated C-peptide correlated with islet number at isolation (P = 0.013) and more strongly with the islet number at transplantation (P = 0.001). In contrast, the correlation of stimulated C-peptide and islet isolation index was weaker (P = 0.018), and this was poorer at transplantation (P = 0.034). Using linear regression, the strongest association with graft function was islet number (r = 0.722, P = 0.001). Islet size was not related to graft function after adjusting for islet volume or number. Conclusions These data show no clear correlation between islet isolation index and graft function; both small and large islets are suitable for transplantation, provided the islets have survived a short culture period postisolation., By analyzing the insulin requirements from 25 islet transplantation recipients, Hughes et al determined the strongest association with graft function was islet number while islet size was not related to graft function after adjusting for islet volume or number.

Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently, the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2 to 3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved.We developed a novel procedure of islet transplantation to the inguinal subcutaneous white adipose tissue (ISWAT) of mice and described functional and morphological characteristics of transplanted syngeneic islets. Also, it was determined whether islet allograft rejection in the ISWAT can be prevented by immunosuppressive agents. Furthermore, it was examined whether human islets function when grafted in this particular site of immune-deficient mice.In this site, transplanted islets are engrafted as clusters and function to reverse streptozotocin-induced diabetes in mice. Importantly, transplanted islets can be visualized by computed tomography and are easily retrievable, and allograft rejection is preventable by blockade of costimulatory signals. Of much importance, the efficiency of islet transplantation in this site is superior to the liver, in which hyperglycemia of diabetic recipient mice is ameliorated after transplantation of 200 syngeneic islets (the islet number yielded from 1 mouse pancreas) to the ISWAT but not to the liver. Furthermore, human islets transplanted in this particular site function to reverse diabetes in immune-deficient mice.Thus, the ISWAT is superior to the liver as the site of islet transplantation, which may lead to improved outcome of clinical islet transplantation.

BACKGROUND The effect of conversion to cytotoxic T lymphocyte-associated protein 4 immunoglobulin (CTLA4Ig) treatment on tacrolimus (TAC)-induced renal dysfunction is well known, but its effect on TAC-induced diabetes mellitus (DM) is still undetermined. In the present study, we tested the diabetogenicity of CTLA4Ig and evaluated the effect of conversion to CTLA4Ig treatment on TAC-induced diabetic rats. METHODS We tested diabetogenicity of CTLA4Ig by escalating doses (0.25, 0.5, 1, 2, and 4 mg/kg weekly) for 4 weeks. In the conversion study, we administered TAC (1.5 mg/kg) for 3 weeks and confirmed TAC-induced DM by intraperitoneal glucose tolerance test. Thereafter, TAC administration was continued, withdrawn, or replaced by CTLA4Ig treatment (1 or 2 mg/kg) for additional 3 weeks. The effect of CTLA4Ig on TAC-induced DM in vivo and in vitro was evaluated by assessing pancreatic islet function, histopathology, oxidative stress, apoptosis, and macrophage infiltration. RESULTS Intraperitoneal glucose tolerance test in the CTLA4Ig groups did not differ from the control group. In addition, plasma insulin level, glucose-induced insulin secretion, and islet viability were not different between the CTLA4Ig and control groups. In the conversion study, TAC withdrawal ameliorated pancreatic islet dysfunction compared with the TAC group, and conversion to CTLA4Ig further improved pancreatic islet function compared with the TAC withdrawal group. TAC-induced oxidative stress, apoptotic cell death, and infiltration of macrophages decreased with TAC withdrawal, and CTLA4Ig conversion further reduced those values. In the in vitro study, CTLA4Ig decreased TAC-induced pancreatic islet cell death and reactive oxygen species production. CONCLUSIONS CTLA4Ig was not diabetogenic, and conversion to CTLA4Ig reduced TAC-induced pancreatic islet injury.

There exists a need for a minimally invasive method of islet transplantation without immunosuppressive drugs for the treatment of type 1 diabetes.In diabetic August Copenhagen Irish rats, an agarose rod containing the cyclic oligopeptide SEK-1005 (agarose-SEK rod) was implanted at 2 dorsal subcutaneous sites. Then these rods were removed, and 1500 islets of Langerhans isolated from Fischer 344 rats were transplanted into each of the pockets.Ten days after implantation of agarose-SEK rods, vascularized pockets were present. Nonfasting blood glucose levels confirmed long-term survival of the allogeneic islet grafts, without immunosuppressive therapy, in 8 of 10 recipients. Flow cytometry and gene expression analyses were performed to investigate the mechanisms underlying graft acceptance. Agarose-SEK rod implantation led to the formation of granulomatous tissue containing regulatory T cells that suppressed immune reactions against the allogeneic islet grafts.These results indicate that the use of an agarose-SEK rod to prevascularize a subcutaneous site may be a useful method for achieving successful allogeneic islet transplantation without immunosuppression.

BACKGROUND Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived β cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. METHODS Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. RESULTS Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic β cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. CONCLUSIONS In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to β cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and β cell function and leads to increased inflammatory signaling.

BACKGROUND Current histological methods cannot accurately determine the survival rate of human pancreatic islets following portal vein infusion. This is due, in part, to the low number of infused islets relative to the whole liver. In this study, we assessed the ability of confocal laser scanning microscopy (CLSM) to track human islets posttransplantation. METHODS Immunodeficient mice were transplanted with human islets. Following engraftment, animals were euthanized, livers procured, and human islet β cells immunofluorescently labeled with an insulin-specific antibody and evaluated by CLSM. A calibration curve comparing the area of insulin + hepatic islet β cells to the number of human islets collected was developed. Levels of human C-peptide were measured in transplant recipients to determine islet function. RESULTS The short-term survival rate of islet transplants was defined as y = 0.0422x + 2.7008, in which x is human islet number and y is liver islet β cell area. Employing CLSM, human islets were detected in immunofluorescent labeled murine liver tissue sections posttransplantation. The β cell-relative area of human islets in 500 islet equivalent (IEQ) specimens was 20.21 ± 1.16 mm and in 1000 IEQ specimens 39.4 ± 2.23 mm posttransplantation. Human islet posttransplant survival rates were 82.9 ± 5.50% (500 IEQ group) and 86.9 ± 5.28% (1000 IEQ group). CONCLUSIONS These data indicate that CLSM can be employed to quantify and characterize pancreatic human islets after transplantation to murine livers.

Background Islet transplantation is a promising option for the treatment of type 1 diabetes. However, the current lack of practical techniques for the isolated islets preservation still hampers the advancement of life-saving islet transplantation. Islet suffers from internal or external stimuli-induced oxidative stress and subsequent inflammation during preservation, which leads to disappointing outcomes regarding islet yield, survival, and function. Reactive oxygen species (ROS) overproduction is the primary cause of oxidative stress that induces islet loss and dysfunction. Thus, in this article, we hypothesized that an endogenous antioxidant, bilirubin, that could efficiently scavenge ROS and inhibit inflammatory reactions could be beneficial for islet preservation. Methods Herein, we studied the effect of bilirubin on the hypothermic preserved (4°C) islets and evaluate the islets viability, insulin secretory function, oxidative stress levels, and in vivo transplantation performance. Results Bilirubin could prevent cellular damages during short-term preservation and maintain the cocultured islets viability and function. The protective role of bilirubin is associated with its antioxidative ability, which dramatically increased the activities of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) and decreased the levels of ROS and malondialdehyde. Diabetic mice transplanted with bilirubin preserved islets were normoglycemic for 28 days, even overmatched the diabetic mouse transplanted with fresh islets. Mice receiving bilirubin cocultured islets required the least time to achieve normoglycemia among all groups and exhibited minimum inflammatory responses during the early transplantation stage. Conclusions By utilizing bilirubin, we achieved highly viable and functional islets after hypothermic preservation to reverse diabetes in mice.

New-onset diabetes is a frequent complication after solid organ transplantation. Although a number of common factors are associated with the disease, including recipient age, body mass index, hepatitis C infection, and use of immunosuppressive drugs, new-onset diabetes after liver transplantation (NODALT) has the following unique aspects and thus needs to be considered its own entity. First, a liver graft becomes the patient's primary metabolic regulator after liver transplantation, but this would not be the case for kidney or other grafts. The metabolic states, as well as the genetics of the graft, play crucial roles in the development of NODALT. Second, dysfunction of the islets of Langerhans is common in cirrhotic patients and would be exacerbated by immunosuppressive agents, particularly calcineurin inhibitors. On the other hand, minimized immunosuppressive protocols have been widely advocated in liver transplantation because of liver tolerance (immune privilege). Third and last, through the "gut-liver axis," graft function is closely linked to gut microbiota, which is now considered an important metabolic organ and known to independently influence the host's metabolic homeostasis. Liver transplant recipients present with specific gut microbiota that may be prone to trigger metabolic disorders. In this review, we proposed 3 possible sites for the origin of NODALT, which are liver, islets, and gut, to help elucidate the underlying mechanism of NODALT.

BACKGROUND Erythropoietin exerts anti-inflammatory, antiapoptotic, and cytoprotective effects in addition to its hematopoietic action. A nonhematopoietic erythropoietin analogue, ARA 290, has similar properties. The efficacy of pancreatic islet transplantation (PITx) is reduced due to islet damage that occurs during isolation and from the severe inflammatory reactions caused by the transplantation procedure. We investigated whether ARA 290 protects islets and ameliorates inflammatory responses following PITx thus improving engraftment. METHODS The effects of ARA 290 on pancreatic islets of C57BL/6J (H-2) mice and on murine macrophages were investigated using an in vitro culture model. As a marginal PITx, 185 islets were transplanted into the liver of streptozotocin-induced diabetic mice (H-2) via the portal vein. Recipients were given ARA 290 (120 μg/kg) intraperitoneally just before and at 0, 6, and 24 hours after PITx. Liver samples were obtained at 12 hours after PITx, and expression levels of proinflammatory cytokines were assessed. RESULTS ARA 290 protected islets from cytokine-induced damage and apoptosis. Secretion of pro-inflammatory cytokines (IL-6, IL-12, and TNF-α) from macrophages was significantly inhibited by ARA 290. After the marginal PITx, ARA 290 treatment significantly improved the blood glucose levels when compared to those of control animals (P < 0.001). Upregulation of monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, IL-1β, and IL-6 messenger RNA expression within the liver was suppressed by ARA 290 treatment. CONCLUSIONS ARA 290 protected pancreatic islets from cytokine-induced damage and apoptosis and ameliorated the inflammatory response after PITx. ARA 290 appears to be a promising candidate for improvement of PITx.

For islet transplantation, pancreas preservation in University of Wisconsin (UW) solution is associated with disadvantages, such as collagenase inhibition, resulting in poor islet yield and islets with poor viability. In this study, we evaluated a novel preservation solution, the extracellular-type c-Jun N-terminal kinase (JNK) inhibitor-containing (EJ) solution.The EJ solution has high sodium-low potassium composition with low viscosity compared to UW solution. Moreover, EJ solution contains a recently developed JNK inhibitor from our laboratory.We first compared the performance of EJ solution with that of UW solution. Islet yield before and after purification was significantly higher in the EJ group than in the UW group. Second, we compared the performance of EJ solution with that of EJ solution without the JNK inhibitor (EJ-J solution). After pancreas preservation in EJ solution, JNK activity was maintained at a relatively low level during islet isolation. Islet yield before and after purification was significantly higher in the EJ group than in the EJ-J group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 61.5% and 7.7% of diabetic mice in the EJ and EJ-J groups, respectively. Moreover, EJ solution exhibited reduced inhibition of collagenase digestion compared with UW solution.Advantages of EJ solution over UW solution were inhibition of JNK activity and reduced collagenase inhibition. EJ solution may therefore be more suitable for islet isolation than UW solution.

There is a strong rationale to pursue the use of neonatal porcine islets (NPIs) as an unlimited source of islets for clinical xenotransplantation. Because NPIs are composed of immature insulin producing beta (ß) cells and ductal precursor cells, they provide an ideal model to examine culture conditions to enhance ß cell proliferation and/or ß cell neoformation from ductal cells. In an attempt to optimize the potential of NPIs as a source of ß cell grafts, we used an in vitro differentiation protocol and measured its effect on the functional maturation and differentiation of NPIs.Pancreata from 1- to 3-day-old neonatal pigs were digested and cultured in standard Ham's F10 media for 5 days. Each independent preparation was then further cultured in Dulbecco's modified Eagle medium nutrient mixture-F12 differentiation media containing growth factors added in a stepwise fashion, or cultured in control Ham's F10 media. After 20 days in culture, islets were assessed for insulin secretory capacity, cellular composition, gene expression, and metabolic activity after transplantation in immunodeficient mice with diabetes.Compared with control islets, differentiated islets exhibited a significantly higher proportion of endocrine cells, proliferating cell nuclear antigen double positive ß cells, and an enhanced glucose-stimulated insulin secretory activity. Mice transplanted with differentiated islets had significantly lower blood glucose values at weeks 18 and 20 compared with nondifferentiated controls and were shown to be more glucose tolerant.Culturing NPIs in a 20-day stepwise differentiation media increases the proportion of endocrine cells and augments both in vitro and in vivo function of the islets.

BACKGROUND During the isolation process, pancreatic islets are exposed to an environment of sterile inflammation resulting in an upregulated inflammatory state before transplantation. Toll-like receptor 4 (TLR4) has been identified as a major mediator of sterile inflammation. Therefore, we sought to determine whether early TLR4 blockade would be effective in reducing the inflammatory burden in islets pretransplant. METHODS Islets from C57BL/6 mice were treated with a TLR4 antagonist during the pancreatic ductal perfusion and digestion steps of the isolation process. Islets were then analyzed for inflammation by RT-PCR and Western blot, and for viability and function in vitro. A syngeneic transplant model using a marginal mass of islets transplanted intraportally into mice with streptozotocin-induced diabetes was used to study transplant outcomes after early TLR4 blockade. RESULTS Diabetic mice receiving 150 islets treated with early TLR4 blockade achieved euglycemia at a higher rate than mice receiving untreated islets (75% vs 29%; P < 0.05) and had improved long-term function (P < 0.05). Serum markers for islet damage and inflammation were significantly reduced posttransplant (P < 0.05). Both the expression of key inflammatory genes and the activation of mitogen-activated protein kinases were reduced by early TLR4 blockade. Islet viability was improved (P < 0.05) while preserving islet insulin secretory capacity postisolation. CONCLUSIONS Early TLR4 blockade protects islets from sterile inflammation-mediated stress sustained during isolation and promotes positive transplant outcomes. Our findings support the use of early TLR4 blockade during clinical islet isolation procedures to reduce pretransplant inflammation and improve transplant outcomes.

Background Islet grafts are transplanted into the liver via a portal vein in 90% of the clinical islet transplantations. However, the portal vein is far from being the ideal infusion site due to its unique drawbacks. These issues necessitated the exploration of an alternatively optimized site for clinical islet transplantation. With the widespread clinical application of percutaneous transhepatic puncture technique, we envisioned the possibility of islet transplantation into the hepatic sinus tract (HST). Methods The HST was created by temporarily placing a medically approved material into the hepatic parenchyma of C57BL/6 mice. The syngeneic islets were transplanted into the HST, after which, the nonfasting blood glucose, intraperitoneal glucose tolerance, and morphology were evaluated. Results A collagen-lined HST was formed by the 28-day implantation of a cylindrical nylon rod. Transplantation of ~300 syngeneic islets into the HST routinely reversed the hyperglycemia of the recipient mice and maintained normoglycemia for longer than 100 days until the graft was removed. The islet grafts within the HST stained positively for insulin, glucagon, and abundant microvessels and achieved comparable results to the islet grafts under the kidney capsule with respect to glycemic control and glucose tolerance. Conclusions These results suggested that an HST can be constructed for islet transplantation by temporarily placing a nylon material in the liver parenchyma. The HST is a promising site for clinical islet transplantation, thereby providing a satisfactory environment for the survival and metabolic function of islet grafts.

T cell-mediated graft rejection is mostly correlated with potent Th1 responses. However, because IFNγ mice reject their graft as efficiently as wild-type (WT) mice, the exact contribution of IFNγ and its transcription factor T-bet remains a matter of debate. Here, we address this question in the context of pancreatic islet allograft to better inform the molecular pathways that hampers islet survival in vivo.Pancreatic islets from BALB/c mice were transplanted in WT, IFNγ, or T-bet C57BL/6 mice. Graft survival and the induction of effector and cytotoxic T-cell responses were monitored.Rejection of fully mismatched islet allografts correlated with high expression of both IFNγ and T-bet in WT recipients. However, allogeneic islets were permanently accepted in T-bet mice, in contrast to IFNγ hosts. Long-term survival correlated with decreased CD4 and CD8 T-cell infiltrates, drastically reduced donor-specific IFNγ and tumor necrosis factor tumor necrosis factor α responses and very low expression of the cytotoxic markers granzyme B, perforin, and FasLigand. In addition, in vitro and in vivo data pointed to an increased susceptibility of T-bet CD8 T cell to apoptosis. These observations were not reported in IFNγ mice, which have set up compensatory effector mechanisms comprising an increased expression of the transcription factor Eomes and cytolytic molecules as well as tumor necrosis factor α-mediated but not IL-4 nor IL-17-mediated allogeneic responses.Anti-islet T-cell responses require T-bet but not IFNγ-dependent programs. Our results provide new clues on the mechanisms dictating islet rejection and may help refine the therapeutic/immunosuppressive regimens applied in diabetic patients receiving islets or pancreas allografts.

Background Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation. Methods Syngeneic mouse islets were transplanted subcutaneously within Sernova Corp's Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance. Results Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels. Conclusions The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies., The authors apply a new implanted subcutaneous cell pouch (CP) device in a mouse diabetes model. Mouse islets transplanted into the CP restore glycemic control with well respond to glucose challenge. CP may serve as a potential alternative to clinical intraportal islet transplantation.

Background Muscle is a promising alternative site for islet transplantation that facilitates rapid restoration of islet vascularization. However, the development of fibrosis suggests massive cellular death after transplantation. This study tested the hypothesis that islet graft function is limited by hypoxia-related death early after intramuscular transplantation, but that this can be overcome by cotransplantation of an oxygen carrier, that is, polymerized bovine hemoglobin (PolyHb). Methods Two hundred islets were transplanted with or without different doses of PolyHb intramuscularly to nondiabetic C57BL/6 and diabetic C57BL/6 nu/nu mice. β-cell hypoxia and apoptosis were evaluated by immunohistochemistry after injection of the biochemical marker pimonidazole or by staining for caspase-3, respectively. Blood glucose concentrations were monitored for 30 days after islet transplantation and animals were then subjected to an intravenous glucose tolerance test. Results Substantial hypoxia was observed in control islet grafts during the first 4 days after transplantation. Cotransplantation of PolyHb resulted in a dose-dependent reduction of β-cell hypoxia, but β-cell apoptosis was only reduced by cotransplantation of low-dose PolyHb (0.03 mg/g body weight) due to the inflammatory effects of higher PolyHb concentrations. Cotransplantation of low-dose PolyHb resulted in improved islet graft function 30 days after transplantation in diabetic mice, with a glucose tolerance comparable to transplantation of 50% more islets. Conclusion We conclude that cotransplantation of islets with PolyHb can be used to effectively bridge the critical hypoxic phase immediately after transplantation, improve islet graft function, and reduce the number of islets needed for successful intramuscular transplantation.

Early and sensitive detection of islet graft damage is essential for improving posttransplant outcomes. MicroRNA 375 (miR375) has been reported as a biomarker of pancreatic β-cell death in small animal models.The miR375 levels were measured in purified human islets, sera from patients with autologous and allogeneic islet transplantation as well as total pancreatectomy alone (nontransplanted group). The miR375 levels were also determined in a miniaturized in vitro tube model comprising human islets and autologous blood.The miR375 expression level in islets was dose-dependent (P0.001) and significantly elevated after islet damage in plasma in the in vitro model (P = 0.003). Clinical analysis revealed that circulating miR375 levels in both autologous and allogeneic islet recipients were significantly elevated for 7 days after islet infusion, compared with the nontransplanted group (P = 0.005 and0.001, respectively). Furthermore, miR375 detected the difference in islet graft damage among 3 different anti-inflammatory protocols for clinical autologous transplantation (P0.01).Circulating miR375 can be a reliable biomarker to detect graft damage in clinical islet transplantation because serum C-peptide and proinsulin levels are difficult to interpret due to the influence of multiple factors, such as β-cell stress and physiological response.

BACKGROUND Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Background It was previously shown that islets are susceptible to oxidative stress due to their inherent low antioxidant capacity. Therefore, in this study, we determined whether treatment of mouse islets with an antioxidant cyanidin-3-O-glucoside (C3G) could enhance their function after transplantation under the kidney capsule or into the portal vein. Methods B6 mouse islets were treated with various concentrations of C3G, their viability, and the expression of antioxidant (HO-1) and antiapoptotic (Bcl-2 and Survivin) genes were determined. The C3G-treated (1.0 μM) or untreated B6 mouse islets (100, 200, and 400 IEQ) were transplanted under the kidney capsule or into the portal vein of diabetic B6 mice, and their blood glucose levels were monitored for more than 100 days after transplantation. Results The C3G-treated islets showed higher cell viability compared to untreated control and the expression of HO-1; Bcl-2 and Survivin genes were enhanced in a concentration-dependent manner. All mice that were transplanted with C3G-treated islets achieved normoglycemia faster than recipients of untreated islets. Mice that received 400, 200, or 100 treated islets transplanted under the kidney capsule achieved normoglycemia, whereas only mice that were transplanted with 400 and 200 treated islets into the portal vein achieved normoglycemia. The mean blood glucose levels of mice that received C3G-treated islets were lower compared to those observed in mouse recipients of untreated islets transplanted under the kidney capsule or into the portal vein. Conclusions Our results show that C3G could enhance the viability of mouse islets and improve their function after transplantation.

Subcutaneous islet transplantation is associated with minimal invasiveness, but poor vascularization. Thus, the optimization of the prevascularization procedures is crucial for improving the outcomes. Although the effectiveness of basic fibroblast growth factor (bFGF) was reported, the optimal procedures remain unclear. We sought to optimize the prevascularization procedures including the use of a novel scaffold, recombinant peptide (RCP).Devices containing various amount of bFGF with/without heparin or RCP were implanted into the subcutaneous space of diabetic C57BL/6 mice. Syngeneic islets were transplanted into the prevascularized space. Blood glucose, intraperitoneal glucose tolerance, and immunohistochemistry were evaluated.The cure rates in all the device groups irrespective of bFGF doses were considerably higher than in the nondevice group. The cure rate in the bFGF0 group was unexpectedly higher than that in the subcutaneous islet transplant alone group (the None group) (57.1% vs 28.6%). Glucose tolerance was ameliorated in the bFGF10(-), 10(+) and 15(-) groups. The number of von Willebrand factor-positive vessels in the bFGF10(+) group was significantly higher than that in the None and bFGF0 groups (P0.01). Taken together, the bFGF10(+) group was considered to have received optimized procedures. In a marginal graft model, the efficiency in the RCP group was better than that in the bFGF10(+) group, furthermore, comparable to that in the intraportal transplantation group. Unlike bFGF, no bleeding and effusion were observed in the RCP group.These results suggest that optimizing biomaterials to induce efficient prevascularization could be a novel strategy for improving subcutaneous islet transplantation.

BACKGROUND The instant blood-mediated inflammatory response (IBMIR) has been shown as a major factor that causes damage to transplanted islets. Withaferin A (WA), an inhibitor of nuclear factor (NF) κB, was shown to suppress the inflammatory response in islets and improve syngeneic islet graft survival in mice. We investigated how treating islets with NF-κB inhibitors affected IBMIR using an in vitro human autologous blood islet model. METHODS Human islets were pretreated with or without NF-κB inhibitors WA or CAY10512 before mixing autologous blood in a miniaturized in vitro tube model. Plasma samples were collected at multiple time points and used for the measurement of C-peptide, proinsulin, thrombin-antithrombin (TAT) complex, and a panel of proinflammatory cytokines. Infiltration of neutrophils into islets was analyzed using immunohistochemistry. RESULTS Rapid release of C-peptide and proinsulin was observed 3 hr after mixing islets and blood in the control group, but not in the NF-κB inhibitor-treated groups, whereas TAT levels were elevated in all three groups with a peak at 6 hr. Significant elevation of proinflammatory cytokines was observed in the control group after 3 hr, but not in the treatment groups. Significant inhibition of neutrophil infiltration was also observed in the WA group compared with the control (P

Background Long-term survival and functions of encapsulated islet grafts need to be evaluated in the absence of immunosuppression. The present study aimed to assess the viability and functions of macroencapsulated islets grafted in nonhuman primates without immunosuppression for 1 year. Methods Islet transplantations were performed in partially pancreatectomized rhesus monkeys (two autologous and four allogenic) without immunosuppression using immunoisolatory devices. Macroencapsulated islets were implanted subcutaneously (5000-8000 IEQ/device) at two sites (left thigh and interscapular region) and were explanted at 2, 6, and 12 months after implantation. Staining for viability and apoptosis, in vivo and in vitro glucose-stimulated insulin release, expression of insulin and glucagon genes, and histopathologic examination of the device were used to assess engraftment potential, viability, and functions of islets. Animals were regularly monitored for dietary intake, body weight, and fasting blood glucose levels after islet transplantation. Results Devices explanted showed vascularization at the end of 2, 6, and 12 months with occasional lymphocytes and minimal fibrosis outside the device. Flow cytometric analysis revealed 97.9%±1.5% and 94.3%±5.71% viable β cells in interscapular site and thigh in autologous recipients and 85.6%±4.01% (interscapular site) and 74.1%±12.05% (thigh) viable β cells in allogenic islet recipients. In vivo glucose challenge test revealed significantly increased glucose-stimulated insulin release (P=0.028) in the left thigh with implant (17.58±3.13 mU/L) compared with the thigh without implant (9.86±1.063 mU/L). Insulin and glucagon gene expression was evident in islets recovered from explanted device. Conclusions These results indicate that subcutaneous implantation of macroencapsulated islets is minimally invasive and has potential for transplantation without immunosuppression.

Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice.Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing.F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P0.05 and P0.05, respectively). Furthermore, F573 improved human islet function when transplanted under the KC (P0.05); whereas F573 did not enhance murine islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P0.05 and P0.05, respectively).The pan-caspase inhibitor F573 markedly reduces human and mouse islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

Background Although islet xenotransplantation is a promising therapy for type 1 diabetes, its clinical application has been hampered by cellular rejection and the requirement for high levels of immunosuppression. The aim of this study was to determine the role of Foxp3 regulatory T (Treg) cells in costimulation blockade-induced dominant tolerance to porcine neonatal islet cell cluster (NICC) xenografts in mice. Methods Porcine-NICC were transplanted under the renal capsule of BALB/c or C57BL/6 recipients and given a single dose of CTLA4-Fc at the time of transplant and 4doses of anti-CD154 mAb to day 6. Depletion of Foxp3Treg cell was performed in DEpletion of REGulatory T cells mice at day 80 posttransplantation. Foxp3Treg cell from spleens of treated BALB/c mice (tolerant Treg cell), and splenocytes were cotransferred into islet transplanted nonobese diabetic background with severe combined immunodeficiency mice to assess suppressive function. Results In treated mice, increased numbers of Foxp3Treg cell were identified in the porcine-NICC xenografts, draining lymph node, and spleen. Porcine-NICC xenografts from treated mice expressed elevated levels of TGF-β, IL-10 and IFN-γ. Porcine-NICC xenograft tolerance was abrogated after depletion of Foxp3Treg cell. Tolerant Treg cell produced high levels of IL-10 and had diverse T cell receptor Vβ repertoires with an oligoclonal expansion in CDR3 of T cell receptor Vβ14. These tolerant Treg cells had the capacity to transfer dominant tolerance and specifically exhibited more potent regulatory function to porcine-NICC xenografts that naive Treg cell. Conclusions This study demonstrated that short-term costimulation blockade-induced dominant tolerance and that Foxp3Treg cell played an essential role in its maintenance. Foxp3Treg cells were activated and had more potent regulatory function in vivo than naive Treg cells.

Background Toll-like receptors (TLRs) are involved in the rejection of solid organ allografts. However, the roles of TLRs in islets are still controversial. We investigated the roles of TLRs in donor islets together with those in recipients in allogeneic islet transplantation. Methods To assess the roles of TLRs in either donor islets or recipients, allogeneic islet transplantation was performed using myeloid differentiation factor 88 (MyD88)-knockout (KO), TLR4-KO, or Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)-KO mice. Results Both polyriboinosinic polyribocytidylic acid and lipopolysaccharide (LPS) stimulation induced the mRNA expression of regulated and normal T cell expressed and secreted, interferon-γ-inducible protein-10, monocyte chemotactic protein-1, interleukin-8, and inducible nitric oxide synthase in murine islets, whereas the induction was attenuated in TRIF-KO, interferon-β promoter stimulator-1-KO, and TLR4-KO mice. When islets from MyD88-KO, TLR4-KO, or TRIF-KO C57BL/6 mice were transplanted to BALB/c recipients, graft survival was not better than that of wild-type (WT) islets. However, the survival of the MyD88-KO islet allograft was significantly prolonged when combined with anti-CD40L. In parallel, LPS stimulation in donor islets interfered with anti-CD40L blockade-mediated long-term survival of islet allografts in TLR4-KO recipients. LPS stimulation increased the perigraft infiltration of both T cells and macrophages. Then again, when islets from WT BALB/c mice were transplanted to MyD88-KO, TRIF-KO, or WT C57BL/6 mice, there was no difference in graft survival, although some of the MyD88-KO recipients obtained long-term graft survival. However, anti-CD40L prolonged graft survival significantly in MyD88-KO recipients. The absence of MyD88 in either donors or recipients decreased the perigraft infiltration of inflammatory cells when combined with anti-CD40L. Conclusions TLRs in both donor islets and recipients are involved in islet allograft rejection.

BACKGROUND The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). METHODS We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. RESULTS The NEM consistently achieved higher islet yields from pancreatitis (P

Background. The ability to predict clinical function of a specific islet batch released for clinical transplantation using standardized variables remains an elusive goal. Methods. Analysis of 10 donor, 7 islet isolation, 3 quality control, and 6 recipient variables was undertaken in 110 islet-after-kidney transplants and correlated to the pre- to 28-day posttransplant change in C-peptide to glucose and creatinine ratio (Delta CP/GCr). Results. Univariate analysis yielded islet volume transplanted (Spearman r = 0.360, P < 0.001) and increment of insulin secretion (r = 0.377, P < 0.001) as variables positively associated to Delta CP/GCr. A negative association to Delta CP/GCr was cold ischemia time (r = -0.330, P < 0.001). A linear, backward-selection multiple regression was used to obtain a model for the transplanted functional islet mass (TFIM). The TFIM model, composed of islet volume transplanted, increment of insulin secretion, cold ischemia time, and exocrine tissue volume transplanted, accounted for 43% of the variance of the clinical outcome in the islet-after-kidney data set. Conclusion. The TFIM provides a straightforward and potent tool to guide the decision to use a specific islet preparation for clinical transplantation.

Background It is known that sirolimus (SRL) aggravates cyclosporine A (CsA)-induced nephrotoxicity and pancreatic injury, but the influence of everolimus (EVR) on CsA-induced organ injury is undetermined. Methods Rats were treated with CsA (15 mg/kg) and EVR or SRL (0.3 mg/kg) subcutaneously for 4 weeks. The influences of EVR or SRL on CsA-induced nephrotoxicity and pancreatic islet dysfunction were compared, and drug interactions between CsA and EVR or SRL were evaluated at blood and tissue levels. Results Treatment with EVR or SRL alone did not cause severe pancreatic dysfunction and renal injury, but when combined with CsA they aggravated CsA-induced pancreatic and renal injury. Drug interactions between CsA and EVR or SRL differed at the tissue level. Combined treatment with CsA and SRL significantly increased the CsA or SRL levels in kidney and pancreas compared with CsA or SRL alone. However, combined treatment with CsA and EVR did not increase CsA or EVR levels in kidney and pancreas. Conclusions Both EVR and SRL aggravate CsA-induced organ injury, but the pharmacologic interaction between EVR and CsA at the tissue level is less than that between CsA and SRL. This finding provides better understanding of the difference between EVR and SRL when combined with CsA treatment.

Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of α-1 antitrypsin (A1AT).Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations. The effect of proteases on ultrastructure of islets and β-cell insulin granules were examined by transmission electron microscopy. The number of insulin granules and insulin-labeled immunogold particles were counted. The in vivo effect of proteases on islet function was studied by transplanting acinar cells adjacent to islet grafts in diabetic mice. The effects of A1AT culture supplementation on protease activity, insulin levels, and islet function were assessed in pure and impure islets.Islet loss after culture was significantly higher in impure relative to pure preparations (30% vs. 14%, P0.04). Lower islet purity was associated with increased protease activity and decreased insulin levels in culture supernatants. Reduced β-cell insulin granules and insulin degradation by proteases were confirmed by transmission electron microscopy. Transplantations in mice showed delayed islet graft function when acinar cells were transplanted adjacent to the islets under the kidney capsule. Supplementation of A1AT to impure islet cultures maintained islet cell mass, restored insulin levels, and preserved islet functional integrity.Culture of impure human islet fractions in the presence of A1AT prevents insulin degradation and improves islet recovery.

Type 1 diabetes (also known as juvenile diabetes) results from the autoimmune destruction of insulin-producing pancreatic β cells, and survival of individuals with the disease depends mainly on daily insulin injections or the use of insulin pumps. Transplantation of donor islets has recently proven effective at restoring insulin production and blood glucose control in type 1 diabetes mellitus patients (1–4). Its widespread application is, however, limited by availability of donor islets, the requirement for long-term use of immunosuppressive drugs to prevent graft rejection, and lower rates of long-term insulin independence (5). Moreover, the reduced islet viability and loss of insulin content that occurs during islet isolation and pre-transplantation culture necessitates the use of large numbers of islets, usually from multiple donors, to achieve insulin independence (6). Identification of factors that can enhance islet survival in vitro and improve islet function post-transplantation will thus help in extending the period of insulin independence in type 1 diabetes patients and reduce the number of donors required for successful islet transplantation. Glial cell line-derived neurotrophic factor (GDNF) is a factor produced by glial cells that plays an important role in the development of the enteric nervous system. GDNF has previously been shown to promote the survival of β-cells in vitro, and to enhance β-cell mass and improve glucose control in mice when transgenically over-expressed under the control of the glial fibrillary acidic protein promoter (2, 7). In this study we investigated the ability of GDNF to improve human islet post-transplant survival and function. Using in vitro and in vivo methods we assessed the effects of culturing human islets in the presence of GDNF on β-cell survival, in vitro insulin secretion, and post transplantation glycemic control. We performed these studies to assess the potential for use of GDNF to enhance human islet viability before transplant in type 1 diabetes patients.

Background Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification. Methods Human islets were isolated from deceased donor (n=30) and chronic pancreatitis pancreata (n=30). Pancreatic tissue densities were measured before purification by the ATGS, and the density gradient range for islet purification in a COBE was customized based on density profiles determined by the ATGS. The efficiency of custom density gradients (CDGs) to recover high islet yield was compared with predefined SDGs. Results Pancreatic tissue densities from autografts were significantly higher than in allograft preparations. In allograft purifications, a higher proportion of islets were recovered using ATGS-guided CDGs (85.9%±18.0%) compared with the SDG method (69.2%±27.0%; P=0.048). Acinar contamination at 60%, 70%, and 80% cumulative islet yield for allografts was significantly lower in the CDG group. In autograft purifications, more islets were recovered with CDGs (81.9%±28.0%) than SDGs (55.8%±22.8%; P=0.03). CDGs effectively reduced islet loss by minimizing islet sedimentation in the COBE bag. Conclusions Using ATGS-guided CDGs maximizes the islet recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation of islets in the COBE bag in autograft preparations.

Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.

Background. After the discontinuation of the manufacturing Liberase HI because of a small potential for prion disease transmission, Roche Diagnostics (Indianapolis, IN) developed a new enzyme product (Liberase MTF [mammalian tissue free]), which is similar to Liberase HI with the exception that no mammalian tissue is used in the manufacture of the collagenase component. We report our experience using the MTF enzyme in clinical islet isolations compared with Serva NB-1 with modified enzyme delivery method. Methods. Islets were isolated from 41 pancreata using MTF enzyme (n= 17) or NB-1 enzyme (n=24). NB-1 enzymes were delivered using a modified (nonsimultaneous) enzyme delivery method whereas isolations using MTF used the standard method of simultaneous collagenase and thermolysin perfusion. Islets were purified on a COBE 2991 Cell Blood Processor and subsequently cultured. Results. The average islet mass after purification was 392±36×10 3 islet equivalent (IE) for MTF versus 371±40×10 3 IE for Serva NB-1 (P=0.63). Post-IE/cm 3 of tissue was 110±9×10 3 IE/cm 3 and 91 ± 11×10 3 IE/cm 3 for MTF and NB-1, respectively (P=0.07). The isolation success rate (>400,000 IE) for MTF was 53% compared with 33% for Serva (P=0.33). Conclusion. We conclude that MTF may be successfully used for high-yield human islet isolation and clinical transplantation and provides similar quality islets to those derived using NB-1.