Encapsulated islet transplantation for patients with type 1 diabetes mellitus

Meirigeng Qi，Shusen Wang，Yong Wang（. Department of Translational Research and Cellular Therapeutics，Diabetes and Metabolic Research Institute，Beckman Research Institute of the City of Hope，Duarte，CA，USA ；2. Organ Transplant Center，Tianjin First Center Hospital，Tianjin 30092，China ；3. Key Laboratory for Critical Care Medicine of the Ministry of Health，Tianjin First Central Hospital，Tianjin 30000，China ；. Division of Transplantation/Department of Surgery，University of Illinois at Chicago，Chicago，IL，USA

【Abstract】 Encapsulation of pancreatic islets has been investigated for over three decades to advance islet transplantation outcomes and to defecate the side effects of immunosuppressive medications. Within the numerous encapsulation systems developed, microencapsulation has been investigated most extensively so far. A lot of materials have been used for microencapsulation in different animal models（including non-human primates or NHPs）and some materials have shown to be of immunoprotection to islet grafts without the need for immunosuppression. In spite of the initial success of microcapsules in NHP models，the combined use of islet transplantation（allograft）and microencapsulation has not yet been successful in clinical trials. This concise review consists of two sections∶Brief summary for transplantation of encapsulated pancreatic islets as a treatment for patients with type 1 diabetes mellitus（T1DM）, and present challenges and future perspectives.

【Key words】 T1DM；Islet transplantation；Microencapsulation

Introduction

Type 1 diabetes mellitus（T1DM），also known as insulin-dependent diabetes mellitus，is an autoimmune disease that causes a progressive destruction of the insulin-producing pancreatic β-cells1-2. As a result，patients require exogenous insulin to maintain normal blood glucose levels. In patients with T1DM，long-termhyperglycemia often causes complications such as nephropathy，neuropathy，and retinopathy. According to a report from the American Diabetes Association（ADA），there are nearly three million children and adults living with T1DM in the U.S. and millions of others affected worldwide3.Management of T1DM and other associated complications are burdensome to both individuals and to society as a whole. Insulin injection is a common method to directly control blood glucose levels. However，intensive insulin therapy can induce more frequent episodes of hypoglycemic symptoms in certain populations of patients with T1DM4-5.Islet transplantation is considered as an improved way to cure T1DM in comparison with insulin injection and whole pancreas transplantation. Absence of insulin in patients with T1DM forces them to use exogenous insulin to maintain normal blood glucose，which can delay or prevent health complications. Theoretically，exogenous insulin can replace β- cells in islets，but practically，the insulin injection cannot maintain stable blood glucose levels. Pancreatic islet transplantation is a procedure to selectively transplant the endocrine part of a whole pancreas（about 2% of the pancreas mass）.In comparison with whole pancreas transplantation，islet transplantation can be conducted via a minimally invasive approach and is associated with minimal or no complications. The islets can be infused via a catheter that has percutaneous portal venous access6.Therefore this procedure can be applied to a wider range of recipients. More importantly，the islet transplantation can provide glycemic control without exogenous insulin and risks of hypoglycemia. The first experimental islet transplantation was conducted in a rodent model in 1972，several years after this a whole pancreas transplantation was initiated in a human patient7. Although islet transplantation has been widely accepted in recent years，the protocol has not obtained a license and is not accepted as a standard clinical treatment. Currently，many islet transplantation centers are planning or initiating license applications for clinical allogeneic islet transplantation.

Transplantation of encapsulated pancreatic islets as a treatment for patients with T1MD

Overview of encapsulation

Cell encapsulation technology is based on the concept of immunoisolation，which was originally presented by Prehn et al. From as early as 1954，Prehn et al.used a type of immunoisolation instrument called the diffusion chamber device8. In that study，the diffusion chamber device was used to prevent the homograft from provoking an immune reaction in the host. Later on this technology was used to protect transplanted cells，known as “artificial cells”9-10. Since islet cells can be isolated and transplanted successfully，the encapsulation technology was soon applied in the field of islet transplantation. Many types of encapsulation technologies have been designed over the last three decades（Figure 1）and investigated in different animals such as mice11，rats12，dogs13-14，and monkeys15-16. These studies demonstrate the feasibility of restoring normoglycemia by implanting allo- and xenografts without immunosuppression. Furthermore，the studies reveal the inconsistency of transplantation outcomes due to differences in encapsulation strategy and in animal models. The studies also suggest that long-term graft survival might depend on enriched and consistent blood supply to the grafts. In the light of the experiences accumulated from the large amount of transplant studies performed in different animal models，scientists and clinicians attempted a trial involving encapsulated allogeneic islet transplantation in patients with T1DM17-20. The following sections address microencapsulation，depict immunology and biocompatibility factors of the devices，outline the approach of local/short-term immunomodulation，and report the trials of clinical encapsulated islet transplantation.

Microencapsulation

The main advantages of the microencapsulation system over other encapsulation technologies are its stable mechanical structure，large surface areato-volume ratio，and improved diffusion profile.Due to the flexible and adjustable characteristics，the microcapsules are mostly fabricated from hydrogels. Over the past 30 years，hydrogel including alginate21，poly（hydroxyethyl methacrylate-methyl methacrylate），agarose22，acrylonitrile copolymers，chitosan23，and polyethelene glycol（PEG）24have been frequently used for microencapsulation. To date，the most preferable material for microencapsulation is alginate. The principle of making microcapsules is based on the envelopment of individual islets in a droplet，which is transformed into a rigid capsule by gelification（in the case of alginate beads）followed by polycation coating（in the case of multiple-layered microcapsules）.

Figure 1 Schematic representation of immunoisolation device or bioartificial pancreas. They can be commonly separated into two categories，intravascular and extravascular device. The latter can further be divided into macroencapsulation and microencapsulation devices.Intravascular and extravascular classifications are based on whether or not it is connected directly to the blood circulation. The macroencapsulation and microencapsulation classifications depend on whether it contains one or more islets in the inside

Alginate，a collective term for a family of polysaccharides synthesized by seaweed and bacteria，is used in a wide range of foods，pharmaceutical products，and other applications25. In molecular terms，alginates are binary linear polysaccharides composed of two monomers，α-L-guluronic（G）and β-D-mannuronic（M）acid，which form M blocks，G blocks，and blocks of alternating sequence（MG）26.In nature，alginates are found to exhibit great variations in composition and arrangement of the two monomers in a polymer chain. Blocks of repeating G units（G blocks）form cavities that bind divalent cations，which cross-link G blocks of other alginate chains27.This in turn allows for the formation of gels as capsules.Hence，G-block sequences are required for the alginate to form a strong gel with divalent ions such as Ca2+，Ba2+and Sr2+. A strong correlation therefore exists between the sequential structure and functional properties of alginates.

To increase the stability and to reduce the permeability of alginate gel beads，a polycation layer is traditionally added to the alginate gel core28-30. However，the successful use of alginate-polycation capsules as carriers for insulin producing cells in vivo has been hampered by the capsule's lack of biocompatibility as well as their mechanical instability. These disadvantages have made controlled insulin release and immunoprotection of islets difficult to achieve.The major obstacle for stability is swelling，causing an increase in pore size and ultimately breakage. This is caused by the loss of calcium from the calcium-alginate gel by e.g. phosphate and citrate，which can bind calcium，and non-gelling ions such as sodium that over time will exchange some of the calcium in the gel31.

Immunology and biocompatibility

Demographics and baseline characteristics within clinical status groups

Immunology studies the host's defense mechanisms against invasion of foreign organisms; either living or non-living. The immune response is often divided into two categories，innate and acquired immune reaction. The innate immune response is non-specific and exists in all individuals. It doesn't distinguish between different organisms and acts rapidly upon the exposure of foreign invaders. The innate immune reaction typically initiates with cellular mediators such as macrophages and neutrophils. The acquired immune reaction is specific and not actively present in all individuals. This specific immune response requires the recognition of a specific antigen by lymphocytes including T and B cells.

Biomaterials are not firmly considered as organisms32，however implantation of biomaterials in a host triggers an immune reaction，which involves many components of the immune system. Biocompatibility is commonly defined as the ability of a biomaterial or other medical device to perform its function properly in a specific application with an appropriate response in the host33-34.“Biocompatibility refers to the ability of a biomaterial to perform its desired function with respect to a medical therapy，without eliciting any undesirable local or systemic effects in the recipients or beneficiary of that therapy，but generating the most appropriate beneficial cellular or tissue response in that specific situation，and optimizing the clinically relevant performance of that therapy”33. The immunoisolation device is not constructed solely by material for the main structure and it also contains islet cells. Therefore，in order for the device to be biocompatible，the bioartificial pancreas must carry out its proper function and it must not harm the host. For an immunoisolation device，biocompatibility has been referred to as the degree of fibrosis after implantation into the host. Recently work has focused on the implantation of microcapsules in larger animals，primarily NHPs，to evaluate the biocompatibility of the microcapsules for clinical islet transplantation.

As noted earlier，alginate is the most commonly used material for islet microencapsulation. The biocompatibility of microcapsules has been tested with the implantation of empty microcapsules in numerous animal models. The peritoneal cavity has been selected as an optimal site for in vivo analysis of microencapsulated islet implantation，as this site can harbor a large volume of microcapsules7. This site is easily accessible during implantation and is relatively safe. It has been reported previously that empty microcapsules，composed of purified alginate，do not elicit any significant foreign body reaction after implantation into the peritoneal cavity of rodents35-36.However，implantation of empty microcapsules into the portal vein of pigs provoke extensive pericapsular cellular overgrowth37. This result indicates that portal vein microcapsule transplantation is incompatible with the current alginate composition.

The evaluation of the function of microencapsulated islets in large animals is a necessary transit point between scientific studies in rodents and its clinical application for humans. Allotransplantation in large animals has been performed to mimic clinical islet transplantation. Soon-Shiong et al. initially reported the long-term reversal of diabetes in dogs using microencapsulated islet allografts38. Recently，allografts in alginate-PLL microcapsules were tested in the absence of anti-rejection medications in pigs but large-scale studies were not documented39.Wang et al. published work on the normalization of blood glucose levels in dogs for up to 214 days with a single transplantation of microencapsulated allogeneic islets without immunosuppressive medication13. Although the NHP is considered as an optimal allotransplantation model，little is published in terms of microencapsulated islet transplantation.In our previous study，we conducted allogeneic islet transplantations in baboons using the modified PMCG microcapsules. Two diabetic baboons were transplanted with an average of 16 475 IEQ/kg encapsulated islets（2-3 transplants）and neither baboon achieved normoglycemia after transplantation. Evenly distributed microcapsules were observed in the peritoneal cavity.Retrieved microcapsules at 4 weeks post-transplant were intact and free of cellular overgrowth around the microcapsules.

Due to the shortage of donor tissues for patients with T1DM，xenotransplantation has drawn the attention of research facilities. Most xenotransplantation uses microencapsulated porcine islets as donor tissue.Sun et al. found that microencapsulated porcine islets transplanted into spontaneously diabetic cynomolgus monkeys survived for 120-800 days with no immunosuppression40. Other groups have tested their encapsulated porcine islets in non-diabetic monkeys15-16. It is notable that all of these transplanted porcine islets were encapsulated in alginate-polycation based microcapsules，which is a microcapsule with less antibody permeability.

In our previous study，human islets encapsulated in Ca2+/Ba2+-alginate microbeads were transplanted into the peritoneal cavity of a diabetic baboon at a dose of 36 000 IEQ/kg. After transplantation，decreased blood glucose and positive C-peptide production were observed up to 2 weeks. Adhesion and clumping of the microcapsules were observed during laparotomy at day 76 post-transplant. Microcapsules that were retrieved at this point presented with fibrotic overgrowth.Xenogeneic tissue can trigger a stronger immune mediated rejection compared to allogeneic tissue，which may explain islet graft dysfunction in this study.Antibody responses against the encapsulated islets were found 20-35 days post-transplant. Similar results were observed in the transplantation of microencapsulated human islets into the peritoneal cavity of diabetic cynomolgus monkey（unpublished data）.

Local or short-term immunomodulation

As mentioned earlier，a variety of natural and synthetic polymers have been used in islet encapsulation.However，inconsistency and poor long-term results have been a major limitation for clinical application.The graft failure is usually initiated by several factors including poor biocompatibility of the implanted materials，hypoxic conditions for islets inside of the capsules，and incomplete immunoprotection41-42. Thus，local or short-term immunomodulation and a non-systematic immunosuppressive treatment have been investigated to improve the encapsulated islet transplant outcomes.

Biocompatibility of capsules is crucial for the long-term survival of the islet graft. Study showed that a 10-day immunosuppressive medication regimen significantly reduced the fibrotic overgrowth around the intraportally implanted empty microcapsules43. Our group also tested the beneficial effects of 2-week long T-cell directed immunosuppressive medication and anti-inflammatory agents（TNF-α blocker）on the biocompatibility of Ca2+/Ba2+-alginate microbeads in cynomolgus monkeys. The results showed that the medications could only prevent fibrotic overgrowth on the surface of the implanted empty microbeads for as long as the medications were administered. This suggests that the extended use of immunosuppressants may have to be administrated to make the Ca2+/Ba2+-alginate microbeads biocompatible，which diminishes the goal of the encapsulation strategy（unpublished data）.

Incomplete immunoprotection is mainly caused by the uncontrollable passage of pro-inflammatory cytokines and other immunoreactive molecules with low molecular weights，such as IL-1β（17.5 KD）and TNF-α（51 KD）through the biopolymer membrane44-45.

Therefore，strategies to block those cytokines have been studied in recent years to improve the graft survival after encapsulated islet transplantation. In a recent study，a peptide inhibitor for the cell surface IL-1 receptor（IL-1R）was conjugated to the hydrogel for capsules to block the interaction between the immobilized cells and the cytokines46. In another strategy，Sertoli cells were used in co-encapsulation with islets cells.These cells are located in the convoluted seminiferous tubules of testes and have been shown to inhibit T and B cell proliferation and IL-2 production47.Co-transplantation of islets with Sertoli cells were shown to have varying protective effects on graft survival in allo-48，concordant（rat to mouse）and discordant（fish to mouse）xeno-49-50，and autoimmune51transplant models. Study showed that the Sertoli cells improve the functional performance of alginate-PLL microencapsulated islets in xenotransplant models（rat-mouse）52. However，this approach has not advanced significantly enough to be used in clinical trials.

Encapsulated islet transplantation in patients with T1DM

Table 1 lists the clinical trials of encapsulated islets transplanted in patients with T1DM. Soon-Shiong et al. reported a successful human encapsulated islet transplant in a diabetic patient who was receiving immunosuppression for a functioning kidney graft17.In the study，a total of 15 000 IEQ/kg alginate-PLL encapsulated islets were implanted intraperitoneally.Insulin independence was demonstrated for 9 months after the procedure，with tight glycemic control noted.Scharp et al. subcutaneously implanted a PAN-PVC macroencapsulation device containing allogeneic islets into 9 patients53. The results concluded that macroencapsulated human islets could survive at the subcutaneous site and that semi-permeable membranes can be designed to protect against both allogeneic immune responses as well as the autoimmune reactions of patients with T1DM.

Calafiore et al. transplanted alginate-PLO microcapsulated islets in a human clinical trial without immunosuppression20. In 2006，the results of the first two patients were published and both patients showed increased C-peptide serum levels，as a measure of islet graft function. Several weeks post transplantation，these two patients presented with an ephemeral incline in exogenous insulin consumption20. In 2011，the same group published the results of encapsulated islet transplantation in 4 patients，which included the follow-up results of the initial two patients reported in 2006 and two other patients transplanted afterwards54.So far，the results from 4 patients have been reported.In all cases the group observed no side effects of the grafting procedure，nor any evidence of immune sensitization. All patients exhibited a lower intake of exogenous insulin，approximately half of the pre-transplantation consumption levels.

Tuch et al. transplanted allogeneic islets encapsulated in Ba2+-alginate microbeads into four diabetic patients without immunosuppression. C-peptide was present on day one after transplantation，but disappeared within a period of one to four weeks. In a recipient of multiple islet infusions，C-peptide was detected at 6 weeks after the third infusion and remained detectable for 30 months. Neither insulin requirement nor glycemic control was altered in any of the patients18.

From 2005 to 2006，two companies，Amcyte，Inc.and Novocell，Inc. announced clinical trials involving encapsulated islet transplantation in patients with T1DM. Amcyte，Inc. planned to conduct clinical trials in twelve patients using islets encapsulated in alginate-PLL microcapsules. These microcapsules were further embedded into a macrocapsule for implantation.Another company，Novocell，Inc.（current name ViaCyte，Inc.），initiated phase 1/2 clinical trials of PEG-encapsulated islet allograft implantation in patients with T1DM. Twelve patients were enrolled in this clinical trial. However，this particular study was terminated. Currently，there is limited information available regarding these two clinical trials.most recent newsletter from the website，a registration study has been launched in 2013 for phase 2b/3 clinical trials，in which 30 patients were enrolled. The LCT product，DIABECEL＠，is expected to be commercially available in 201619.

Most recently，Jacobs-Tulleneers-Thevissen et al.published work on transplantation of Ca2+/Ba2+-alginate microbeads containing allogeneic islets in a patient56. The alginate microbeads were harvested 3 months after transplantation and were conglomerated in the peritoneal cavity. Another report announced a commercial product of the macroencapsulation device called the Cell Pouch SystemTM. This device can be subcutaneously implanted. The device has a unique ability of releasing anti-rejection drugs locally. The Cell Pouch SystemTMis currently preparing for clinical trials.

The future perspectives

At present，there is a large amount of islet encapsulation-related research in progress around the world trying to eliminate the use of immunosuppressants in patients with T1DM. This research is largely uncoordinated and a well-documented systematic analysis of the various capsule types has not been completed. The correlation between NHPs and human subjects in biocompatibility of device and function of transplanted islets is poorly demonstrated.Despite the numerous clinical trials conducted by academic institutes and biotechnological companies，encapsulated islet transplantation has not been perfected18-20，54，56. With regards to the mixed set of results，there are three main factors limiting the progression of microencapsulated islet transplantation towards clinical application. First，the variability of raw materials in the manufacturing process has impeded the development of a reliable microencapsulation system. Second，current biocompatibility testing relies heavily on in vivo rodent models，which does not

Table 1 Encapsulated islet transplantation in patients with T1DM

Xenotransplantation has attracted much attention in the field of islet transplantation. In the light of such consideration，transplantation of microencapsulated xenogeneic islets，especially porcine islets，has commenced in patients with T1DM. In 1996，Living Cell Technologies（LCT），a company based in New Zealand，initiated a clinical trial involving encapsulated porcine islet transplantation. In this trial，porcine islets were encapsulated in alginate-PLO microcapsules and implanted into the peritoneal cavity of patients without immunosuppression. Nine and a half years after transplantation，laparotomy of one of the patients showed the presence of microcapsules in the peritoneal cavity，some of which still contained live pig islet cells. However，the majority of cells appeared to be necrotic55. As of now，the company reported in their website that a total of 14 patients with T1DM were enrolled in the phase 1/2 clinical trial of DIABECEL＠conducted in New Zealand and Russia19. The first four patients received approximately 10 000 IEQ/kg encapsulated islets and showed an average reduction of 76% in episodes of clinically significant hypoglycemia unawareness after 30-52 weeks of follow-up. Four patients from each of the second and third groups received 15 000 and 20 000 IEQ/kg of encapsulated islets respectively and the follow-up of these particular patients is ongoing. The last two patients have received a dose at 5 000 IEQ/kg and were enrolled to construct the dose ranging data needed to determine a target product profile for phase 3 clinical trials. Based on the strongly support patients with T1DM. Finally，there is a significant inconsistency in results observed among individual laboratories even with the use of similar biomaterials and experimental approaches.

Taking all these obstacles into account，the development of a centralized in vitro and in vivo testing center in the future would allow for a more comprehensive，consistent，and species-specific examination of biocompatibility for the encapsulation system. A collaborative consortium may need to be organized，which should lead to the standardization in material selections，techniques，animal models，and procedures. Under active collaboration between research facilities，the end goal of providing islet encapsulation as a viable cure for patients with T1DM without immunosuppressant would be achievable.

Acknowledgements

The author would like to thank Dr. Igor Lacik and Dr.Berit L. Strand for sharing their knowledge of islet encapsulation and Dr. James McGarrigle for reviewing and editing the manuscript.