Human islet transplantation can provide good glycemic control in diabetic recipients without exogenous insulin. However, a major factor limiting its application is the recipient's need to adhere to life-long immunosuppression, something that has serious side effects. Microencapsulating human islets is a strategy that should prevent rejection of the grafted tissue without the need for anti-rejection drugs. Despite promising studies in various animal models, the encapsulated human islets so far have not made an impact in the clinical setting. Many non-immunological and immunological factors such as biocompatibility, reduced immunoprotection, hypoxia, pericapsular fibrotic overgrowth, effects of the encapsulation process and post-transplant inflammation hamper the successful application of this promising technology. In this review, strategies are discussed to overcome the above-mentioned factors and to enhance the survival and function of encapsulated insulin-producing cells, whether in islets or surrogate β-cells. Studies at our center show that barium alginate microcapsules are biocompatible in rodents, but not in humans, raising concerns over the use of rodents to predict outcomes. Studies at our center also show that the encapsulation process had little or no effect on the cellular transcriptome of human islets and on their ability to function either in vitro or in vivo. New approaches incorporating further modifications to the microcapsule surface to prevent fibrotic overgrowth are vital, if encapsulated human islets or β-cell surrogates are to become a viable therapy option for type 1 diabetes in humans.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated islets. A strategy to combat PFO is the use of mesenchymal stem cells (MSC). MSC have anti-inflammatory properties and their potential can be enhanced by stimulation with proinflammatory cytokines. This study investigated whether co-encapsulation or co-transplantation of MSC with encapsulated islets would reduce PFO and improve graft survival. Stimulating MSC with a cytokine cocktail of IFN-γ and TNF-α enhanced their immunosuppressive potential by increasing nitric oxide production and secreting higher levels of immunomodulatory cytokines. In vitro, co-encapsulation with MSC did not affect islet viability but significantly enhanced glucose-induced insulin secretion. In vivo, normoglycemia was achieved in 100% mice receiving islets co-encapsulated with stimulated MSC as opposed to 71.4% receiving unstimulated MSC and only 9.1% receiving encapsulated islets alone. Microcapsules retrieved from both unstimulated and stimulated MSC groups had significantly less PFO with improved islet viability and function compared to encapsulated islets alone. Levels of peritoneal immunomodulatory cytokines IL-4, IL-6, IL-10 and G-CSF were significantly higher in MSC co-encapsulated groups. Similar results were obtained when encapsulated islets and MSC were co-transplanted. In summary, co-encapsulation or co-transplantation of MSC with encapsulated islets reduced PFO and improved the functional outcome of allotransplants.

Transplantation of pancreatic islets encapsulated within immuno-protective microcapsules is a strategy that has the potential to overcome graft rejection without the need for toxic immunosuppressive medication. However, despite promising preclinical studies, clinical trials using encapsulated islets have lacked long-term efficacy, and although generally considered clinically safe, have not been encouraging overall. One of the major factors limiting the long-term function of encapsulated islets is the host's immunological reaction to the transplanted graft which is often manifested as pericapsular fibrotic overgrowth (PFO). PFO forms a barrier on the capsule surface that prevents the ingress of oxygen and nutrients leading to islet cell starvation, hypoxia and death. The mechanism of PFO formation is still not elucidated fully and studies using a pig model have tried to understand the host immune response to empty alginate microcapsules. In this review, the varied strategies to overcome or reduce PFO are discussed, including alginate purification, altering microcapsule geometry, modifying alginate chemical composition, co-encapsulation with immunomodulatory cells, administration of pharmacological agents, and alternative transplantation sites. Nanoencapsulation technologies, such as conformal and layer-by-layer coating technologies, as well as nanofiber, thin-film nanoporous devices, and silicone based NanoGland devices are also addressed. Finally, this review outlines recent progress in imaging technologies to track encapsulated cells, as well as promising perspectives concerning the production of insulin-producing cells from stem cells for encapsulation.

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 above strategies are likely to enhance the outcomes of clinical islet transplants.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated pancreatic islets. Modification of the microcapsule membrane aimed at preventing PFO should improve graft survival. This study investigated the effect of macromolecular Corline Heparin Conjugate (CHC) binding on intrinsic properties of alginate microcapsules and assessed the anti-fibrotic potential of this strategy both in vitro and in vivo. CHC was bound to alginate microcapsules using a layer-by-layer approach incorporating avidin. CHC binding to alginate microcapsule was visualized by confocal microscopy. Effects of CHC binding on microcapsule size, strength, and permeability were assessed, and the anti-clotting activity of bound CHC was determined by coagulation assay. Effect of CHC binding on the viability of encapsulated human islets was assessed in vitro, and their ability to function was assessed both in vitro and in vivo in diabetic immunodeficient mice. The potential of bound CHC to reduce PFO was assessed in vivo in different rat transplantation models. Confocal microscopy demonstrated a uniform coating of CHC onto the surface of microcapsules. CHC binding affected neither size nor permeability but significantly increased the tensile strength of alginate microcapsules by ~1.3-fold. The bound CHC molecules were stable and retained their anti-clotting activity for 3 weeks in culture. CHC binding affected neither viability nor function of the encapsulated human islets in vitro. In vivo CHC binding did not compromise islet function, and diabetes was reversed in all recipients with mice exhibiting lower blood glucose levels similar to controls in oral glucose tolerance tests. CHC binding was beneficial and significantly reduced PFO in both syngeneic and allogeneic rat transplantation models by ~65% and ~43%, respectively. In conclusion, our results show a new method to successfully coat CHC on alginate microcapsules and demonstrate its beneficial effect in increasing capsule strength and reduce PFO. This strategy has the potential to improve graft survival of encapsulated human islets.

As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 lM desferrioxamine (DFO) prior to transplantation. Cell viability, glucoseinduced insulin secretion, VEGF (Vascular endothelial growth factor), HIF-1a (Hypoxia inducible factor-1 alpha), caspase-3 and caspase-8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase-3 and caspase-8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6-and 2.5-fold at 24 and 72 h, respectively, with a concomitant increase in HIF-1a levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF-1a and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.

Xenotransplantation of microencapsulated fetal pig islet-like cell clusters (FP ICCs) offers a potential cellular therapy for type 1 diabetes. Although microcapsules prevent direct contact of the host immune system with the xenografted tissue, poor graft survival is still an issue. This study aimed to characterise the nature of the host immune cells present on the engrafted microcapsules and effects on encapsulated FP ICCs that were transplanted into immunocompetent mice. Encapsulated FP ICCs were transplanted into the peritoneal cavity of C57BL/6 mice. Grafts retrieved at days 1, 3, 7, 14 and 21 post-transplantation were analysed for pericapsular fibrotic overgrowth (PFO), cell viability, intragraft porcine gene expression, macrophages, myofibroblasts and intraperitoneal murine cytokines. Graft function was assessed ex vivo by insulin secretion studies. Xenogeneic immune response to encapsulated FP ICCs was associated with enhanced intragraft mRNA expression of porcine antigens MIP-1α, IL-8, HMGB1 and HSP90 seen within the first two weeks post-transplantation. This was associated with the recruitment of host macrophages, infiltration of myofibroblasts and collagen deposition leading to PFO which was evident from day 7 post-transplantation. This was accompanied by a decrease in cell viability and loss of FP ICC architecture. The only pro-inflammatory cytokine detected in the murine peritoneal flushing was TNF-α with levels peaking at day 7 post transplantation. This correlated with the onset of PFO at day 7 implying activated macrophages as its source. The anti-inflammatory cytokines detected were IL-5 and IL-4 with levels peaking at days 1 and 7, respectively. Porcine C-peptide was undetectable at all time points post-transplantation. PFO was absent and murine intraperitoneal cytokines were undetectable when empty microcapsules were transplanted. In conclusion, this study demonstrated that the macrophages are direct effectors of the xenogeneic immune response to encapsulated FP ICCs leading to PFO mediated by a combination of both pro- and anti-inflammatory cytokines.