Islet Encapsulation Research Articles

Encapsulation of protein microfiber networks supporting pancreatic islets

Networks of discrete, genipin-crosslinked gelatin microfibers enveloping pancreatic islets were incorporated within barium alginate microcapsules. This novel technique enabled encapsulation of cellular aggregates in a spherical fibrous matrix <300 μm in diameter. Microfibers were produced by vortex-drawn extrusion within an alginate support matrix. Optimization culminated in a hydrated fiber diameter of 22.3 ± 0.4 μm, a significant reduction relative to that available through current gelatin microfiber spinning techniques, while making the process more reliable and less labor intensive. Microfibers were encapsulated at 40 vol % within 294 ± 4 μm 1.6% barium alginate microparticles by electrostatic-mediated dropwise extrusion. Pancreatic islets extracted from Sprague Dawley rats were encapsulated within the microparticles and analyzed over 21 days. Acridine orange and propidium iodide fluorescent viability staining and light microscopy indicated a significant increase in viability for islets within the fiber-embedded particles relative to fiber-free controls at days 7, 14, and 21. The fiber-embedded system also promoted cellular aggregate cohesion, reducing the incidence of dispersed islet morphologies within the capsules from 31 to 8% at day 21. Further enquiry into benefits of islet encapsulation within a protein fiber network will be the subject of future investigation.

Enhanced function of pancreatic islets co-encapsulated with ECM proteins and mesenchymal stromal cells in a silk hydrogel

Pancreatic islet encapsulation within biosynthetic materials has had limited clinical success due to loss of islet function and cell death. As an alternative encapsulation material, a silk-based scaffold was developed to reestablish the islet microenvironment lost during cell isolation. Islets were encapsulated with ECM proteins (laminin and collagen IV) and mesenchymal stromal cells (MSCs), known to have immunomodulatory properties or to enhance islet cell graft survival and function. After a 7 day in vitro encapsulation, islets remained viable and maintained insulin secretion in response to glucose stimulation. Islets encapsulated with collagen IV, or laminin had increased insulin secretion at day 2 and day 7, respectively. A 3.2-fold synergistic improvement in islet insulin secretion was observed when islets were co-encapsulated with MSCs and ECM proteins. Furthermore, encapsulated islets had increased gene expression of functional genes; insulin I, insulin II, glucagon, somatostatin, and PDX-1, and lower expression of the de-differentiation genes cytokeratin 19 and vimentin compared to non-encapsulated cells. This work demonstrates that encapsulation in silk with both MSCs and ECM proteins enhances islet function and with further development may have potential as a suitable platform for islet delivery in vivo.

Exendin-4 Increases the Expression of Hypoxia-Inducible Factor-1α in Rat Islets and Preserves the Endocrine Cell Volume of Both Free and Macroencapsulated Islet Grafts

In this study, we evaluated the effects of exendin-4 on free and encapsulated islet grafts in a rodent model. We also investigated the role of a transcription factor, hypoxia-inducible factor-1 (HIF-1), in mediating the beneficial effects of exendin-4. Diabetic athymic mice were transplanted with free rat islets under the kidney capsule or with macroencapsulated rat islets SC with or without exendin-4, islet preculture (exendin-4 0.1 nM for 20 h), and/or recipient treatment (IP 100 ng/day, day 0-7). The mice were followed for 4 weeks and the graft function and β-cell volume were evaluated. The effects of exendin-4 on islet HIF-1α mRNA and protein expression and on ATP content in a rat insulinoma cell line (INS-1E) were also examined. Preculture with exendin-4 followed by recipient treatment improved the outcome of both free (73% graft function vs. 26% in controls, p = 0.03) and macroencapsulated islet grafts (100% vs. 25% in controls, p = 0.02). In macroencapsulated grafts, the exendin-4-treated group had significantly larger endocrine volume, less graft necrosis, and more blood vessels around the capsule. In rat islets cultured with exendin-4, HIF-1α mRNA and protein expression were significantly enhanced. ATP content was increased in exendin-4-treated INS-1E cells under hypoxic conditions. The improved functional outcome after transplantation of a marginal islet mass with a brief initial treatment with exendin-4 is related to a larger surviving endocrine cell volume. Exendin-4 may improve islet graft resistance to hypoxia during the peritransplant period by increasing the expression of HIF-1α.

Long-Term Viability of Transplanted Hybrid Cellular Spheroids within Chondrocyte Sheets

Encapsulation of transplanted cells within an immunoisolating membrane may provide a new strategy for protecting these cells from recipient immune responses without the use of immunosuppressive drugs. We have previously reported a novel concept of immunoisolation and immunodelusion using recipient cells instead of traditional artificial materials. We developed a chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas (CSI-BAP) that would enable transplantation of cells across allogeneic and xenogeneic barriers without the cells being recognized as donor cells and without the need for immunosuppression. Recently, we have constructed hybrid cellular spheroids (HCSs) containing cells from two different cell lines (RIN-5F, an insulin-secreting cell line, and Hep-G2, a hepatocellular carcinoma cell line) to enhance the function and biocompatibility of the HCSs. These HCSs were then encapsulated with multiple layers of chondrocyte sheets obtained from the auricular cartilage of Sprague-Dawley (SD) rats. The in vitro ability of the CSI-BAP to secrete insulin was tested before transplantation. Histological evaluation of CSI-BAP chondrocyte microencapsulated immunoisolated islet morphology and viability of allogeneic or xenogeneic cell lines was performed 100 days after the CSI-BAP was transplanted into SD rats. Morphological evaluations revealed good viability of the islets and progression of islet encapsulation. In vitro insulin secretion from the CSI-BAP was well maintained. Additionally, insulin and albumin secretion from the CSI-BAP was confirmed by in vivo immunohistochemical examination. Moreover, the cell lines transplanted into the subcutaneous space in the form of HCSs within the chondrocyte sheets showed good viability of more than 100 days and sustained insulin and albumin secreting ability.

Stability of alginate microbead properties in vitro

Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3 weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy.

Nano-scale encapsulation enhances allograft survival and function of islets transplanted in a mouse model of diabetes.

The success of islet transplantation as a treatment for type 1 diabetes is currently hampered by post-transplantation loss of functional islets through adverse immune and non-immune reactions. We aimed to test whether early islet loss can be limited and transplant survival improved by the application of conformal nano-coating layers to islets. Our novel coating protocol used alternate layers of phosphorylcholine-derived polysaccharides (chitosan or chondroitin-4-sulphate) and alginate as coating materials, with the binding based on electrostatic complexation. The in vitro function of encapsulated mouse islets was studied by analysing islet secretory function and cell viability. The in vivo function was evaluated using syngeneic and allogeneic transplantation in the streptozotocin-induced mouse model of diabetes. Nano-scale encapsulated islets retained appropriate islet secretory function in vitro and were less susceptible to complement- and cytokine-induced apoptosis than non-encapsulated control islets. In in vivo experiments using a syngeneic mouse transplantation model, no deleterious responses to the coatings were observed in host animals, and the encapsulated islet grafts were effective in reversing hyperglycaemia. Allo-transplantation of the nano-coated islets resulted in preserved islet function post-implantation in five of seven mice throughout the 1month monitoring period. Nano-scale encapsulation offers localised immune protection for implanted islets, and may be able to limit early allograft loss and extend survival of transplanted islets. This versatile coating scheme has the potential to be integrated with tolerance induction mechanisms, thereby achieving long-term success in islet transplantation.

Encapsulated islets transplantation: Past, present and future

Islet transplantation could become an ideal treatment for severe diabetes to prevent hypoglycemia shock and irreversible diabetic complications, once some of the major and unresolved obstacles are overcome, including limited donor supplies and side effects caused by permanent immunosuppressant use. Approximately 30 years ago, some groups succeeded in improving the blood glucose of diabetic animals by transplanting encapsulated islets with semi-permeable membranes consisting of polymer. A semi-permeable membrane protects both the inner islets from mechanical stress and the recipient's immune system (both cellular and humoral immunities), while allowing bidirectional diffusion of nutrients, oxygen, glucose, hormones and wastes, i.e., immune-isolation. This device, which enables immune-isolation, is called encapsulated islets or bio-artificial pancreas. Encapsulation with a semi-permeable membrane can provide some advantages: (1) this device protects transplanted cells from the recipient's immunity even if the xenogeneic islets (from large animals such as pig) or insulin-producing cells are derived from cells that have the potential for differentiation (some kinds of stem cells). In other words, the encapsulation technique can resolve the problem of limited donor supplies; and (2) encapsulation can reduce or prevent chronic administration of immunosuppressants and, therefore, important side effects otherwise induced by immunosuppressants. And now, many novel encapsulated islet systems have been developed and are being prepared for testing in a clinical setting.

In situ formation and collagen-alginate composite encapsulation of pancreatic islet spheroids

In this study, we suggest in situ islet spheroid formation and encapsulation on a single platform without replating as a method for producing mono-disperse spheroids and minimizing damage to spheroids during encapsulation. Using this approach, the size of spheroid can be controlled by modulating the size of the concave well. Here, we used 300 μm concave wells to reduce spheroid size and thereby eliminating the central necrosis caused by large volume. As the encapsulation material, we used alginate and collagen-alginate composite (CAC), and evaluated their suitability through diverse in vitro tests, including measurements of viability, oxygen consumption rate (OCR), hypoxic damage to encapsulated spheroids, and insulin secretion. For in situ encapsulation, alginate or CAC was spread over a concave microwell array containing spheroids, and CaCl 2 solution was diffused through a nano-porous dialysis membrane to achieve uniform polymerization, forming convex structures. By this process, the formation of uniform-size islet spheroids and their encapsulation without an intervening replating step was successfully performed. As a control, intact islets were evaluated concurrently. The in vitro test demonstrated excellent performance of CAC-encapsulated spheroids, and on the basis of these results, we transplanted the islet spheroids-encapsulated with CAC into the intraperitoneal cavity of mice with induced diabetes for 4 weeks, and evaluated subsequent glucose control. Intact islets were also transplanted as control to investigate the effect of encapsulation. Transplanted CAC-encapsulated islet spheroids maintained glucose levels below 200 mg/dL for 4 weeks, at which they were still active. At the end of the implantation experiment, we carried out intraperitoneal glucose tolerance test (IPGTT) in mice to investigate whether the implanted islets remained responsive to glucose. The glucose level in mice with CAC-encapsulated islet spheroids dropped below 200 mg/dL 60 min after glucose injection and was stably maintained. In conclusion, the proposed encapsulation method enhances the viability and function of islet spheroids, and protects these spheroids from immune attack.

New Alginate Microcapsule System for Angiogenic Protein Delivery and Immunoisolation of Islets for Transplantation in the Rat Omentum Pouch

Severe hypoxia caused by a lack of vascular supply and an inability to retrieve encapsulated islets transplanted in the peritoneal cavity for biopsy and subsequent evaluation are obstacles to clinical application of encapsulation strategies for islet transplantation. We recently proposed an omentum pouch model as an alternative site of encapsulated islet transplantation and have also described a multi-layer microcapsule system suitable for coencapsulation of islets with angiogenic protein in which the latter could be encapsulated in an external layer to induce vascularization of the encapsulated islet graft. The purpose of the present study was to determine the angiogenic efficacy of fibroblast growth factor (FGF-1) released from the external layer of the new capsule system in the omentum pouch graft. We prepared 2 groups of alginate microspheres, each measuring ∼600 μm in diameter with a semipermeable poly-L-ornithine (PLO) membrane separating 2 alginate layers. While one group of microcapsules contained no protein (control), FGF-1 (1.794 μg/100 microcapsules) was encapsulated in the external layer of the other (test) group. From each of the 2 groups, 100 microcapsules were transplanted separately in an omentum pouch created in each normal Lewis rat and were retrieved after 14 days for analysis of vessel density using the technique of serial sample sections stained for CD31 with quantitative three-dimensional imaging. We found that FGF-1 released from the external layer of the test microcapsules induced a mean ± SD vessel density (mm 2) of 198.8 ± 59.2 compared with a density of 128.9 ± 10.9 in pouches measured in control capsule implants ( P = .03; n = 5 animals/group). We concluded that the external layer of our new alginate microcapsule system is an effective drug delivery device for enhancement of graft neovascularization in a retrievable omentum pouch.

Function and Viability of Human Islets Encapsulated in Alginate Sheets: In Vitro and in Vivo Culture

Islet encapsulation offers an immune system barrier for islet transplantation, and encapsulation within an alginate sheetlike structure offers the ability to be retrievable after transplanted. This study aims to show that human islets encapsulated into islet sheets remain functional and viable after 8 weeks in culture or when transplanted into the subcutaneous space of rats. Human islets were isolated from cadaveric organs. Dissociation and purification were done using enzymatic digestion and a continuous Ficoll-UWD gradient. Purified human islets were encapsulated in alginate sheets. Human Islet sheets were either kept in culture, at 37°C and 5% CO 2, or transplanted subcutaneously into Lewis rats. After 1, 2, 4, and 8 weeks, the human islet sheets were retrieved from the rats and assessed. The viability of the sheets was measured using fluorescein diacetate (FDA)/propidium iodide (PI), and function was measured through glucose-stimulated insulin release, in which the sheets were incubated for an hour in low-glucose concentration (2.8 mmol/L) and then high (28 mmol/L), then high (28 mmol/L) plus 3-isobutyl-1-methylxanthine (50 μm). Human islet sheets remained both viable, above 70%, and functional, with a stimulation index (insulin secretion in high glucose divided by insulin secretion in low glucose) above 1.5, over 8 weeks of culture or subcutaneous transplantation. Islet transplantation continues to make advances in the treatment of type 1 diabetes. These preliminary results suggest that encapsulated islets sheets can survive and maintain islet viability and function in vivo, within the subcutaneous region.

The Midas Touch

A gold:gadolinium label can be used to track encapsulated pancreatic islets after transplantation.

Effect of alginate encapsulation on the cellular transcriptome of human islets

Encapsulation of human islets may prevent their immune rejection when transplanted into diabetic recipients. To assist in understanding why clinical outcomes with encapsulated islets were not ideal, we examined the effect of encapsulation on their global gene (mRNA) and selected miRNAs (non-coding (nc)RNA) expression. For functional studies, encapsulated islets were transplanted into peritoneal cavity of diabetic NOD-SCID mice. Genomics analysis and transplantation studies demonstrate that islet origin and isolation centres are a major source of variation in islet quality. In contrast, tissue culture and the encapsulation process had only a minimal effect, and did not affect islet viability. Microarray analysis showed that as few as 29 genes were up-regulated and 2 genes down-regulated (cut-off threshold 0.1) by encapsulation. Ingenuity analysis showed that up-regulated genes were involved mostly in inflammation, especially chemotaxis, and vascularisation. However, protein expression of these factors was not altered by encapsulation, raising doubts about the biosignificance of the gene changes. Encapsulation had no effect on levels of islet miRNAs. In vivo studies indicate differences among the centres in the quality of the islets isolated. We conclude that microencapsulation of human islets with barium alginate has little effect on their transcriptome.

Effect of prolonged gelling time on the intrinsic properties of barium alginate microcapsules and its biocompatibility

Pericapsular fibrotic overgrowth (PFO) may be attributed to an immune response against microcapsules themselves or to antigen shedding through microcapsule pores from encapsulated islet tissue. Modification of microcapsules aimed at reducing pore size should prevent PFO and improve graft survival. This study investigated the effect of increased gelling time (20 vs. 2 min) in barium chloride on intrinsic properties of alginate microcapsules and tested their biocompatibility in vivo. Prolonged gelling time affected neither permeability nor size of the microcapsules. However, prolonged gelling time for 20 min produced brittle microcapsules compared to 2 min during compression test. Encapsulation of human islets in both types of microcapsules affected neither islet viability nor function. The presence of PFO when transplanted into a large animal model such as baboon and its absence in small animal models such as rodents suggest that the host immune response towards alginate microcapsules is species rather than alginate specific.

The impact of hyperglycemia and the presence of encapsulated islets on oxygenation within a bioartificial pancreas in the presence of mesenchymal stem cells in a diabetic Wistar rat model

This study investigates the potential of bone marrow (BM-MSCs) versus adipose mesenchymal stem cells (AMSCs) to potentiate the oxygenation of encapsulated islets in a subcutaneous bioartificial pancreas. Oxygen pressures (inside subcutaneous implants) were followed in vivo (by electronic paramagnetic resonance) in non-diabetic/diabetic rats transplanted with encapsulated porcine islets or empty implants up to 4 weeks post-transplantation. After graft explantation, neoangiogenesis surrounding the implants was assessed by histomorphometry. Angiogenic properties of BM-MSCs and AMSCs were first assessed in vitro by incubation of the cells in hypoxia chambers, under normoxic/hypoxic and hypo-/hyperglycemic conditions, followed by quantification of vascular endothelial growth factor (VEGF) release. Second, the in vivo aspect was studied by subcutaneous transplantation of encapsulated BM-MSCs and AMSCs in diabetic rats and assessment of the cells’ angiogenic properties as described above. Diabetic state and islet encapsulation induced a significant decrease of oxygenation of the subcutaneous implant and an increased number of cells expressing VEGF. AMSCs demonstrated a significantly higher VEGF secretion than BM-MSCs in vitro. In vivo, AMSCs improved the implant’s oxygenation and vascularization. Diabetes and islet encapsulation significantly reduced the oxygenation of a subcutaneous bioartificial pancreas. AMSCs can improve oxygenation by VEGF release in hypoxia and hyperglycemia states.

Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice

BackgroundType 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants.Methodology/Principal FindingsIn the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3–4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach.Conclusionsh-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes.

Xenotransplantation: from the lab to the clinic

This manuscript presents a summary of the Symposium "Xenotransplantation, from the Lab to the Clinic," held at the XXIII International Congress of The Transplantation Society, August 2010. This Symposium was timely in view of recent developments in translational research in the field, and the initiation of clinical trials after a thorough review by regulatory authorities and scientific experts. Three experts presented an update on the status of preclinical research with the perspective of moving forward to clinical trials; first data from clinical trials using encapsulated porcine islet cells; and the regulatory aspects for clinical application of a xenotransplantation product.

Encapsulation of Langerhans' islets: Microtechnological developments for transplantation

AbstractThere is an increasing trend to apply microsystems and microfluidics to solve medical and biomedical tasks. Microfluidic modules are used to modify and manipulate cells and cell clusters for therapeutic applications. Specifically, a method and technical system for encapsulation of Langerhans' islets as an option for the future treatment of diabetes mellitus is described. Type‐1 diabetes patients suffer from an absolute lack of the hormone insulin caused by an autoimmune process destroying the Langerhans' islets. One way to restore glucose‐dependent insulin secretion is the transplantation of human pancreatic islet cells (85% beta cells) from cadaveric donors. However, to prevent the rejection of the transplanted cells by the immune system of the host, a life long immunosuppressive medication is necessary. One possible way to prevent this rejection would be the transplantation of immunoseparated Langerhans' islets, which are encapsulated in a 3D‐alginate matrix polymerized with ions like Ba2+. As has been successfully shown in animal experiments, this technique would furthermore allow the transplantation of xenograft islets and thus help to solve the problem of shortage in donor organs. We present a microfluidics‐based system for encapsulation of cells in alginate.

Functional Capacity of Human Islets After Long-Distance Shipment and Encapsulation

Human islets produced at an isolation center are shipped to researchers, usually taking short periods to arrive at their destination. In this study, we investigated whether human islets after long-distance shipment across the Pacific Ocean for 2 to 3 days and encapsulation could maintain their functionality. Human islets were encapsulated in alginate and viability assessed using carboxyfluorescein diacetate and propidium iodide. Stimulation index after static glucose incubation was calculated. Streptozotocin-induced diabetic nonobese diabetic/severe combined immunodeficient mice were transplanted with 3000, 2000, or 1000 islet equivalents of nonencapsulated and encapsulated islets and glucose levels monitored. When levels were normal, the graft was retrieved and assessed. Viability of human islets was unaltered after long-distance shipment with a retrieval rate of 88.3% ± 1.9%. After encapsulation, the viability was unchanged (before encapsulation 86.1% ± 0.7% vs after encapsulation 80.8 ± 0.7%) at 11 days after isolation. Function in vitro of nonencapsulated and encapsulated islets was unaffected with a stimulation index of 2.2 and 1.9, respectively. Euglycemia was achieved in 100% mice receiving 2000 and 3000 islet equivalents of nonencapsulated and encapsulated islets, respectively. Capsules retrieved after transplantation was intact, free floating, and contained viable islets. Human islets can be shipped safely for long distances without compromising viability and function even after encapsulation and culture.

Science to Practice: Versatile Method to Track Transplanted Encapsulated Islet Cells with Multiple Imaging Modalities

Pancreatic islet transplantation may provide an effective therapy for patients with type I diabetes. By encapsulating islets in alginate capsules, their survival is substantially improved. Methods to monitor the distribution of the encapsulated islets during delivery and afterward would help to optimize this type of therapy. Barnett and coworkers (1) showed, for the first time, it is possible to visualize encapsulated islets by using magnetic resonance (MR) imaging, computed tomography (CT), and ultrasonography (US) after the inclusion of perfluorocarbon in the capsules. Importantly, it was shown that the therapeutic effect of the islets was sustained for more than 50 days when implanted into mice.

Long-term graft function of cryostored alginate encapsulated rat islets

Microencapsulation of pancreatic islets before transplantation is a promising approach to enable graft function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenic islet tissue is a major problem. One concept to overcome these shortcomings is the cryopreservation of encapsulated allogenic islets. Recently, we reported a gentle cryopreservation protocol for rat islets encapsulated in an alginate-based microcapsule system. Here, we report for the first time long-term transplantation data of these cryopreserved microencapsulated islets. We detected a stable graft function for more than 12 month (experiments still continuing) after transplantation of 2500 cryopreserved microencapsulated CD rat islets in streptozotocin-diabetic Wistar rats. Moreover, the glucose clearance rate during an IPGTT was well preserved up to 56 weeks after transplantation. In addition, hyperglycemic blood glucose levels after removal of rat islet grafts 12 and 56 weeks after transplantation confirmed the efficacy of the encapsulated islets. Finally, the retrieved encapsulated rat islets responded well with a 7-fold increase of insulin secretion to a glucose stimulus (12 and 56 weeks). In conclusion, our study demonstrates for the first time that cryopreservation of encapsulated rat islets is possible without substantial losses on graft function for a very long time.