PEGDA microencapsulated allogeneic islets reverse canine diabetes without immunosuppression.

BACKGROUND: Protection of islets without systemic immunosuppression has been a long-sought goal in the islet transplant field. We conducted a pilot biocompatibility/safety study in healthy dogs followed by a dose-finding efficacy study in diabetic dogs using polyethylene glycol diacrylate (PEGDA) microencapsulated allogeneic canine islets. METHODS: Prior to the transplants, characterization of the canine islets included the calculations determining the average cell number/islet equivalent. Following measurements of purity, insulin secretion, and insulin, DNA and ATP content, the islets were encapsulated and transplanted interperitoneally into dogs via a catheter, which predominantly attached to the omentum. In the healthy dogs, half of the microspheres injected contained canine islets, the other half of the omentum received empty PEGDA microspheres. RESULTS: In the biocompatibility study, healthy dogs received increasing doses of cells up to 1.7 M cells/kg body weight, yet no hypoglycemic events were recorded and the dogs presented with no adverse events. At necropsy the microspheres were identified and described as clear with attachment to the omentum. Several of the blood chemistry values that were abnormal prior to the transplants normalized after the transplant. The same observation was made for the diabetic dogs that received higher doses of canine islets. In all diabetic dogs, the insulin required to attempt to control blood glucose was cut by 50-100% after the transplant, down to no required insulin for the course of the 60-day study. The dogs had no adverse events and behavioral monitoring suggested normal activity after recovery from the transplant. CONCLUSIONS AND IMPLICATIONS: The study provides evidence that PEGDA microencapsulated canine islets reversed the signs of diabetes without immunosuppression and led to states of insulin-independence or significantly lowered insulin requirements in the recipients.

Improved yield of canine islet isolation from deceased donors.

BACKGROUND: Canine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes. RESULTS: Here, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group). CONCLUSIONS: In summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

Integration of mesenchymal stem cells into islet cell spheroids improves long-term viability, but not islet function.

Pancreatic islets, especially the large islets (> 150µm in diameter) have poor survival rates in culture. Co-culturing with mesenchymal stem cells (MSCs) has been shown to improve islet survival and function. However, most co-culture studies have been comprised of MSC surrounding islets in the media. The purpose of this study was to determine whether islet survival and function was improved when the 2 populations of cells were intermingled with each other in a defined geometry. Hybrid spheroids containing 25, 50 or 75 or 90% islets cells with appropriate numbers of MSCs were created along with spheroids comprised of only islet cells or only MSCs. Spheroids were tested for yield, viability, diameter, cellular composition, and glucose-stimulated insulin secretion. The 25% islet/75% MSC group created the fewest spheroids, with the poorest survival and insulin secretion and the largest diameter. The remaining groups were highly viable with average diameters under 80µm at formation. However, the hybrid spheroid groups preferred to cluster in islet-only spheroids. The 50, 75 and 90% islet cell groups had excellent long-term survival with 90-95% viability at 2 weeks in culture, compared with the islet only group that were below 80% viability. The glucose-stimulated insulin secretion was not statistically different for the 50, 75, or 90 groups when exposed to 2.4, 16.8, or 22.4 mM glucose. Only the spheroids with 25% islet cells had a statistically lower levels of insulin release, and the 100% had statistically higher levels at 22.4 mM glucose and in response to secretagogue. Thus, imbedded co-culture improved long-term viability, but failed to enhance glucose-stimulated insulin secretion in vitro.

Long-term cryopreservation of reaggregated pancreatic islets resulting in successful transplantation in rats.

Preservation of pancreatic islets for long-term storage of islets used for transplantation or research has long been a goal. Unfortunately, few studies on long-term islet cryopreservation (1 month and longer) have reported positive outcomes in terms of islet yield, survival and function. In general, single cells have been shown to tolerate the cryopreservation procedure better than tissues/multicellular structures like islets. Thus, we optimized a method to cryopreserve single islet cells and, after thawing, reaggregated them into islet spheroids. Cryopreserved (CP) single human islet cells formed spheroids efficiently within 3-5 days after thawing. Approximately 79% of islet cells were recovered following the single-cell cryopreservation protocol. Viability after long-term cryopreservation (4 weeks or more) was significantly higher in the CP islet cell spheroids (97.4 ± 0.4%) compared to CP native islets (14.6 ± 0.4%). Moreover, CP islet cell spheroids had excellent viability even after weeks in culture (88.5 ± 1.6%). Metabolic activity was 4-5 times higher in CP islet cell spheroids than CP native islets at 24 and 48 h after thawing. Diabetic rats transplanted with CP islet cell spheroids were normoglycemic for 10 months, identical to diabetic rats transplanted with fresh islets. However, the animals receiving fresh islets required a higher volume of transplanted tissue to achieve normoglycemia compared to those transplanted with CP islet cell spheroids. By cryopreserving single cells instead of intact islets, we achieved highly viable and functional islets after thawing that required lower tissue volumes to reverse diabetes in rats.

A simple, reliable method for high-throughput screening for diabetes drugs using 3D ß-cell spheroids.

Early screens for new diabetes drugs rely on monolayers of ß-cells, which are known to be poor predictors of the in vivo response. Previously, we developed a method to create uniform islet spheroids from freshly-dispersed human donor tissue for drug screening. While the human engineered islets worked well to reduce donor-to-donor variability, it is difficult and expensive to obtain sufficient high-quality human islets for drug testing. Thus, this study utilized a genetically-modified ß-cell culture line (INS-1832/13) in 2D and as 3D spheroids and compared the results to human islet tissue formed into spheroids using a high-throughput 384-well format. In response to increasing concentrations of glucose, all 3 groups increased insulin release, but the cultured ß-cells (2D and 3D) were more sensitive to glucose (EC50 5.85mM for 2D ß-cells, 16.24mM for 3D ß-cell spheroids) than the human islet spheroids (EC50 53.69mM). The order of responses to glybenclamide was human spheroids >3D ß-cell culture >2D ß-cell culture. In response to caffeine, the ß-cells in 2D or 3D were more responsive compared to the human islet spheroids (EC50 0.39 and 0.31mM for 2D and 3D ß-cells respectively). When exposed to inhibitors of insulin secretion (nifedipine and diazoxide), the responses were more similar between groups. Z' calculations, indicative of assay quality, determined that the 3D ß-cell spheroids reached the criteria of an excellent to ideal drug screen assay more consistently than the other test models. In conclusion, 3D ß-cell spheroids from a cultured cell line can be used in HTS assays that, according to reference drugs tested here, are sensitive and predictive of the in vivo response.

Long-term liraglutide treatment is associated with increased insulin content and secretion in ß-cells, and a loss of α-cells in ZDF rats.

The ultimate treatment goal of diabetes is to preserve and restore islet cell function. Treatment of certain diabetic animal models with incretins has been reported to preserve and possibly enhance islet function and promote islet cell growth. The studies reported here detail islet cell anatomy in animals chronically treated with the incretin analog, liraglutide. Our aim was to quantitatively and qualitatively analyze islet cells from diabetic animals treated with vehicle (control) or liraglutide to determine whether normal islet cell anatomy is maintained or enhanced with pharmaceutical treatment. We harvested pancreata from liraglutide and vehicle-treated Zucker Diabetic Fatty (ZDF) rats to examine islet structure and function and obtain isolated islets. Twelve-week-old male rats were assigned to 3 groups: (1) liraglutide-treated diabetic, (2) vehicle-treated diabetic, and (3) lean non-diabetic. Liraglutide was given SC twice daily for 9 weeks. As expected, liraglutide treatment reduced body weight by 15% compared to the vehicle-treated animals, eventually to levels that were not different from lean controls. At the termination of the study, blood glucose was significantly less in the liraglutide-treated rats compared to vehicle treated controls (485.8±22.5 and 547.2±33.1mg/dl, respectively). Insulin content/islet (measured by immunohistochemistry) was 34.2±0.7 pixel units in vehicle-treated rats, and 54.9±0.6 in the liraglutide-treated animals. Glucose-stimulated insulin secretion from isolated islets (measured as the stimulation index) was maintained in the liraglutide-treated rats, but not in the vehicle-treated. However, liraglutide did not preserve normal islet architecture. There was a decrease in the glucagon-positive area/islet and in the α-cell numbers/area with liraglutide treatment (6.5 cells/field), compared to vehicle (17.9 cells/field). There was an increase in ß-cell numbers, the ß- to α-cell ratio that was statistically higher in the liraglutide-treated rats (24.3±4.4) compared to vehicle (9.1±2.8). Disrupted mitochondria were more commonly observed in the α-cells (51.9±10.3% of cells) than in the ß-cells (27.2±4.4%) in the liraglutide-treated group. While liraglutide enhanced or maintained growth and function of certain islet cells, the overall ratio of α- to ß-cells was decreased and there was an absolute reduction in islet α-cell content. There was selective disruption of intracellular α-cell organelles, representing an uncoupling of the bihormonal islet signaling that is required for normal metabolic regulation. The relevance of the findings to long-term liraglutide treatment in people with diabetes is unknown and should be investigated in appropriately designed clinical studies.

Small human islets comprised of more ß-cells with higher insulin content than large islets.

For the past 30 years, data have suggested that unique islet populations exist, based on morphology and glucose sensitivity. Yet little has been done to determine the mechanism of these functional differences. The purpose of this study was to determine whether human islets were comprised functionally unique populations, and to elucidate a possible mechanism. Islets or pancreatic sections from 29 human donors were analyzed. Islets were isolated and measured for insulin secretion, cell composition and organization, insulin and glucagon granule density and insulin content. Insulin secretion was significantly greater in small compared with large islets. In sectioned human pancreata, ß-cells comprised a higher proportion of the total endocrine cells in small islets (63%) than large islets (39%). A higher percentage of ß-cells in small islets contacted blood vessels (44%) compared with large islets (31%). Total insulin content of isolated human islets was significantly greater in the small (1323 ± 512 µIU/IE) compared with large islets (126 ± 48 µIU/IE). There was less immunostaining for insulin in the large islets from human pancreatic sections, especially in the core of the islet, compared with small islets. The results suggest that differences in insulin secretion between large and small islets may be due to a higher percentage of ß-cells in small islets with more ß-cells in contact with blood vessels and a higher concentration of insulin/ß-cell in small islets.

Engineering islets for improved performance by optimized reaggregation in a micromold.

Isolated islets can provide a source of tissue for research, transplantation, and drug discovery to develop therapies for diabetes. Empirical modeling of islet diffusion barriers demonstrated that only the outermost layers of cells were exposed to glucose and sufficient oxygen levels, resulting in core cell death. Islets under a diameter of 100 µm exhibited a lower diffusion barrier, superior survival rates, and improved functional properties. Utilizing these observations, we engineered optimal islets by dispersing them into single cells and reaggregating them over several days in a micromold. These custom-designed micromolds contained conical-shaped recesses that enhanced reaggregation of cells into a defined geometry. The engineered islets, or Kanslets, were all under 100 µm in diameter, and had the same general cellular composition as native islets. Kanslets continued to produce new insulin molecules and had microvilli on the islet surface, much like native islets. The engineered islets had a statistically higher viability (percent of live cells), and increased glucose diffusion compared to native islets. In addition, they remained responsive to varying glucose levels by secreting insulin. When transplanted into diabetic rats, engineered islets performed reduced random blood glucose to normal levels within 48 h. Optimally, engineering islets may be a suitable alternative to utilizing native, isolated islet tissue for a variety of applications. Reaggregating tissue in an optimized manner using our engineered micromold approach has immense impact for three-dimensional tissue production and its subsequent use in research, drug discovery, and the clinic.

KU-32, a novel drug for diabetic neuropathy, is safe for human islets and improves in vitro insulin secretion and viability.

KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on the in vitro viability and function of human islets. Treating human islets with KU-32 for 24 hours showed no toxicity as assessed using the alamarBlue assay. Confocal microscopy confirmed that with a minimum of 2-day exposure, KU-32 improved cellular viability by blocking apoptosis. Functionally, isolated human islets released more glucose-stimulated insulin when preincubated in KU-32. However, diabetic BKS-db/db mice, a model for type 2 diabetes, administered KU-32 for 10 weeks did not show any significant changes in blood glucose and insulin levels, despite having greater insulin staining/beta cell in the pancreas compared to untreated BKS db/db mice. In summary, KU-32 did not harm isolated human islets and may even be protective. However, the effect does not appear significant enough to alter the in vivo metabolic parameters of diabetic mice.

Low insulin content of large islet population is present in situ and in isolated islets.

The existence of morphologically distinct populations of islets in the pancreas was described over 60 years ago. Unfortunately, little attention has been paid to possible functional differences between islet subpopulations until recently. We demonstrated that one population, the small islets, were superior to large islets in a number of functional aspects. However, that work did not determine whether these differences were inherent, or whether they arose because of the challenge of isolation procedures. Nor, were there data to explain the differences in insulin secretion. We utilized immunohistochemistry, immunofluorescence, ELISA, and transmission electron microscopy to compare the unique characteristics found in isolated rat islet populations in situ and after isolation. Insulin secretion of small isolated islets was significantly higher compared to large islets, which correlated with higher insulin content/area in small islets (in situ), a higher density of insulin secretory granules, and greater insulin content/volume in isolated islets. Specifically, the core b-cells of the large islets contained less insulin/cell with a lower insulin granule density than peripheral b-cells. When insulin secretion was normalized for total insulin content, large and small islets released the same percentage of total insulin. Small islets had a higher density of cells/area than large islets in vitro and in situ. The data provide a possible explanation for the inferior insulin secretion from large islets, as they have a lower total cell density and the b-cells of the core contain less insulin/cell.

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome.

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter >150 µm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter <100 µm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 µm/min in small islets and 2.8 µm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150 µm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.

Adhesion of pancreatic beta cells to biopolymer films.

Dramatic reversal of Type 1 diabetes in patients receiving pancreatic islet transplants continues to prompt vigorous research concerning the basic mechanisms underlying patient turnaround. At the most fundamental level, transplanted islets must maintain viability and function in vitro and in vivo and should be protected from host immune rejection. Our previous reports showed enhancement of islet viability and insulin secretion per tissue mass for small islets (<125 mum) as compared with large islets (>125 mum), thus, demonstrating the effect of enhancing the mass transport of islets (i.e. increasing tissue surface area to volume ratio). Here, we report the facile dispersion of rat islets into individual cells that are layered onto the surface of a biopolymer film towards the ultimate goal of improving mass transport in islet tissue. The tightly packed structure of intact islets was disrupted by incubating in calcium-free media resulting in fragmented islets, which were further dispersed into individual or small groups of cells by using a low concentration of papain. The dispersed cells were screened for adhesion to a range of biopolymers and the nature of cell adhesion was characterized for selected groups by quantifying adherent cells, measuring the surface area coverage of the cells, and immunolabeling cells for adhesion proteins interacting with selected biopolymers. Finally, beta cells in suspension were centrifuged to form controlled numbers of cell layers on films for future work determining the mass transport limitations in the adhered tissue constructs. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 676-685, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.