Evolution of Islet Transplantation for the Last 30 Years.

REVIEW

Evolution of Islet Transplantation for the Last 30 Years Alan C. Farney, MD, PhD,* David E. R. Sutherland, MD, PhD,† and Emmanuel C. Opara, PhD‡ Abstract: In this article, we will review the changes that have occurred in islet transplantation at the birth of Pancreas 30 years ago. The first attempts at β-cell replacement in humans, pancreas and islet transplantation, were performed in the 1960s and 1970s. Although pancreas transplantation has been an accepted treatment for severe labile diabetes predating the emergence of the journal, allogeneic islet transplantation remains experimental. Current investigations within islet transplantation focus to improve islet function after transplantation. Improving islet viability during isolation, exploring ways to increase engraftment, and protection from the host immune system are some of the goals of these investigative efforts. The major barriers to clinical islet transplantation are shortage of human pancreas, the need for immunosuppression, and the inadequacy of the islet isolation process. It is generally accepted that islet encapsulation is an immunoisolation tool with good potential to address the first 2 of those barriers. We have therefore devoted a major part of this review to the critical factors needed to make it a clinical reality. With improved islet isolation techniques and determination of the best site of engraftment as well as improved encapsulation techniques, we hope that islet transplantation could someday achieve routine clinical use. Key Words: islet isolation, microencapsulation, transplantation, diabetes (Pancreas 2016;45: 8–20)

HISTORICAL PERSPECTIVES Insulin Therapy A little more than 60 years before the initial publication of Pancreas, Banting, Best, Colip, and Macleod discovered a pancreatic “principle” responsible for glucose homeostasis. Banting and Macleod shared the Nobel prize for the discovery of insulin with their colleagues.1 The discovery of insulin converted an often rapidly fatal disease (particularly for patients with the clinical equivalent of type 1 diabetes) to a chronic condition requiring lifelong treatment. At the time, many thought that the ability to administer insulin exogenously would prove to cure diabetes, but blood glucose control was soon found to be a lifelong commitment and long-term imperfections in glycemic control were subsequently associated with the so-called secondary complications of diabetes (diabetic nephropathy, retinopathy, neuropathy, and vascular disease). Improved control of diabetes reduces the risk of secondary complications,2 but secondary complications of diabetes continue to significantly diminish life expectancy and quality of life in many patients with diabetes. Exogenous insulin administration remains the standard method to clinically control high blood glucose levels in insulin-

From the *Department of General Surgery, Wake Forest Institute of Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC; †Department of Surgery, University of Minnesota School of Medicine, Minneapolis, MN; and ‡Wake Forest Institute of Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC. Received for publication December 29, 2014; accepted May 28, 2015. Reprints: Emmanuel C. Opara, PhD, Wake Forest Institute of Regenerative Medicine, Wake Forest School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157 (e‐mail: [email protected]). The authors declare no conflict of interest. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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requiring diabetic patients. The development of long-acting insulin, such as glargine (Lantus), has improved glucose management for many patients. The pancreas, of course, does not make longacting insulin, but its physiologic responses are sustained, making the use of either an insulin pump or long-acting insulin necessary to attempt to achieve similar glucose control in the clinical setting. Some patients manage blood glucose by continuous shortacting insulin administration via pump, augmenting the dose supplied by the pump at mealtime. An insulin pump is essentially an elementary artificial pancreas. A more advanced artificial pancreas that could better mimic the function of the endocrine pancreas would couple a glucose sensor with an implantable insulin pump.3 Although insulin pumps improve blood glucose control, their cost is prohibitive for many patients. By default, the use of long-acting insulin supplemented with shortacting insulin administration at mealtimes, referred to as “basal bolus” management, is the most commonly employed means of exogenous insulin therapy. In this approach, the amount of carbohydrate consumed (estimated in grams) is used to determine the dose of insulin necessary to control blood glucose after a meal. Ideally, patients check finger stick blood glucose levels before meals and at bedtime and administer insulin injections at each of these time points. Blood glucose monitoring is similarly onerous for those patients using insulin pumps, impacting quality of life and making full compliance difficult. In addition, further attempts to improve blood glucose control often increase the risk of hypoglycemia.4 If close enough was good enough, then current methods of insulin administration would suffice, but long-term results from the Diabetes Control and Complications Trial have shown that the threshold level of risk for secondary complications of diabetes is normal glucose homeostasis.2 Currently, only transplantation of pancreatic endocrine tissue, either a whole organ pancreas or islets, is able to consistently achieve this ultimate goal.

Endocrine Replacement Therapy Kelly et al5 performed the first clinical pancreas transplant at the University of Minnesota in 1966. Pancreas transplantation re-establishes endogenous insulin secretion that is responsive to normal feedback regulation, a clinical result that currently cannot and may never be entirely achieved by artificial means. Since 1966, more than 30,000 pancreas transplants have been performed worldwide. According to the Scientific Registry of Transplant Recipients, the 1-year rate of pancreas graft survival is 90% when a pancreas and a kidney are transplanted simultaneously (SPK), 83% when pancreas is transplanted after kidney (PAK), and 80% when pancreas is transplanted alone.6 Most pancreatic grafts are from deceased donors, although transplantation of a segment of the pancreas donated by a living donor is possible and available at a few centers.7 Whole organ pancreas transplantation requires the initial surgical implantation and lifelong immunosuppression to prevent graft rejection. A common perception is that the surgical morbidity of pancreas transplantation is high. Whole organ pancreas transplantation is a major operation requiring laparotomy, major vascular exposure and anastomosis, and management of pancreatic exocrine secretions usually by duodenoenterostomy or urinary bladder drainage. Surgical complications, such as graft thrombosis, result in early pancreas graft loss in up to 5% to 7%,8 but the main risk of pancreas transplantation Pancreas • Volume 45, Number 1, January 2016

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Pancreas • Volume 45, Number 1, January 2016

is related to immunosuppression to prevent rejection. Type 1 diabetic patients who receive a living donor pancreas from an identical twin do not require immunosuppression to prevent rejection but must take it to prevent recurrent autoimmunity.9 Autoimmune recurrence is uncommon among pancreas recipients if immunosuppression is used. Most pancreas transplants are performed with immunosuppression induction therapy (usually monoclonal or polyclonal T-cell–depleting antibody) and maintenance immunosuppression with a calcineurin inhibitor (cyclosporine or tacrolimus), an antimetabolite (mycophenolic acid) plus or minus corticosteroids.10 Owing to the limited availability of human pancreases and the need for immunosuppression, relatively few pancreas transplants are done compared with the entire diabetic population. Improvements in surgical technique or immunotherapy are unlikely to make whole organ pancreas transplantation available to most patients with diabetes. Islet tissue has the ability to engraft at a variety of ectopic sites (outside of the pancreas) as a “free” nonvascularized tissue graft. Experimentally, the liver, spleen, kidney capsule, brain, testis, thymus, and intraperitoneal sites represent a partial list of murine and rat tissues that allow engraftment of islets.11–17 In humans, the clinically preferred site is the liver, although other sites have been used.18 Except in the case of surgical diabetes, where a patient also has exocrine insufficiency, transplantation of only the pancreatic islets meets the clinical need of the

Progress and Challenges in Islet Transplantation

diabetic patient and avoids the technical complications associated with whole organ pancreas transplant. Islets are transplanted by transfusion into the portal vein and embolization into the liver. The transplanted islets engraft in the distal portal triad (Fig. 1). Compared with whole organ transplantation, islet transplantation is technically easier and the recovery is quicker. However, the islet transplant procedure is not without the risk of complications. Intraportal embolization of islet tissue, particularly unpurified islet tissue, has resulted in hepatic infarction, disseminated intravascular coagulation, and portal thrombosis in human subjects.20,21 Transmission of adventitious infection is possible. With the use current good manufacturing process isolation techniques and purification, the risk of infusion-related complications is relatively low but not zero. Similar to whole organ transplantation, the main risk of allogeneic islet transplantation is the need for immunosuppression. There is no objective evidence that islet transplantation requires less immunosuppression than whole organ pancreas transplantation, so the overall risk for each procedure is roughly the same. The efficiency of islet recovery from the whole organ pancreas and the susceptibility of allogeneic islets to immune attack (both alloimmunity and autoimmunity) are challenges to successful clinical islet transplantation. Of the approximately 1 million islets in an adult human pancreas, only half or fewer are successfully isolated on a consistent basis.22,23 As a result, clinical islet

FIGURE 1. Illustration of islet transplantation (with permission from Serup and Madsen19). © 2015 Wolters Kluwer Health, Inc. All rights reserved.



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transplantation often requires 2 or more donor pancreases to provide a sufficient number of isolated islets to achieve insulin independence.24 Because islet isolation requires manipulation of human tissue, the process must be carried out in a good manufacturing process facility, which adds to the expense of the procedure. Although an National Institutes of Health-funded phase 3 trial of islet transplantation is nearing completion, islet transplantation in humans remains “experimental” from the point of view of the Food & Drugs Administration and health insurance.25 For all of the previous reasons, clinical islet transplantation lags far behind whole organ pancreas transplantation in terms of numbers of transplants performed and accessibility for patients with severe labile diabetes. Nonetheless, islet transplantation, as compared with whole organ pancreas transplant, has greater potential to address challenges posed by immunity and the shortage of donor organs. Whereas whole organ pancreas transplantation is nearly maximized as far as a therapeutic option for diabetes, islet transplantation, as a cell therapy, allows greater flexibility in terms of regeneration, manipulation, and immunoisolation. The section on islet microencapsulation discusses some of these possibilities.

STATUS OF ISLET TRANSPLANTATION BEFORE PANCREAS The islets of Langerhans or only approximately 2% of the pancreas regulates glucose metabolism. Separation of the islets from the exocrine pancreas, a necessary step for islet transplantation for diabetes, has evolved over time. Microdissection, a technique used in the 1950s and earlier, produced only limited amounts of islet tissue, insufficient for transplantation but adequate for some physiological studies. In the 1960s, Moskalewski26 employed collagenase to free islets from the surrounding exocrine tissue. This process became more efficient, allowing isolation of up to hundreds of islets, when collagenase was perfused into the pancreatic duct, as described by Lacy and Kostianovsky.27 After this modification, islet isolation, at least in rodents, could provide enough islets to develop islet transplant models. Using streptozotocin to induce diabetes, Ballinger and Lacy12 isolated normal islets from an inbred strain of rats and transplanted the islets to the peritoneum of diabetic rats of the same strain, thereby restoring glucose control. Of the same laboratory, Scharp et al28 described purification of islets using ficoll density gradients and transplantation of the islets into the portal vein of the liver. Studies in large animal models soon followed. Using nonpurified, collagenase-dispersed pancreatic tissue, Mirkovitch and Campiche29 transplanted autologous islets into the spleens of dogs and, along with others, confirmed that both autologous and allogeneic islet transplantation was conceptually feasible in large animals.30,31 In humans, successful allogeneic islet transplantation was elusive; the only “bright” spot being autologous islet transplantation for patients who required pancreatectomy for benign disease of the pancreas. In 1980, Sutherland et al32 published the University of Minnesota experience with islet transplantation in humans: allogeneic islet transplants in 7 diabetic patients who were already on immunosuppression for a previous renal allograft (islet after kidney transplant) and autologous islet transplantation in 3 patients with chronic pancreatitis who had pancreatectomy. Of the 7 allogeneic islet grafts, none were clinically successful (all continued to require insulin administration), although there was evidence of C-peptide production in some of the patients. In contrast, of the 3 autologous islet transplants, 2 remained insulin free for more than a year after pancreatectomy, demonstrating the feasibility of islet transplantation in humans.32

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A PubMed “snap shot” of the literature just before the birth of Pancreas shows the islet transplant field actively investigating methodological improvements in islet isolation,33 optimal sites for implantation of islets,17,34–37 immunoprotection,11,38–40 and the immunology of allogeneic and autoimmune destruction of islet grafts.41–48 Although much of this work used small animal models, mainly mice and rats, some investigators were beginning to pursue studies in large animals, particularly in dogs, but also primates.49 Large animal work, though, remained limited by the inability to consistently obtain large numbers of purified islets and the lack of effective immunosuppression regimens. In 1984, Gray et al33 described a process for human islet isolation including pancreatic duct collagenase injection, mechanical separation, and nylon mesh filtration, followed by ficoll density gradient purification. Most laboratories used a similar approach for islet isolation in large animals, of which the canine model was most amenable30 but porcine and other large animal models remained nearly impossible. Therefore, elucidation of mechanisms of islet rejection and means to prevent rejection were largely carried out in rodent models of islet transplantation, where sufficient numbers of islets could be isolated to carry out studies with numbers sufficient to allow statistical analysis. UV irradiation of isolated islets, anti-Ia serum, antibodies to dendritic cells, cyclosporine, and periods of islet culture were found to prolong islet graft survival in mice and rats.17,42,43,48,50 In some of these models, long-term graft acceptance occurred; the strategies were thought to induce a form of tolerance or immune nonresponsiveness to the islet grafts in these alloimmune, but not autoimmune, small animal models.51 Immunological isolation was studied as an alternative means to protect grafted islets from the host immune system. In 1984, Tze and Tai35 carried out intracerebral islet transplants in the biobreeding rat, demonstrating that the brain was a protected immune site and that islets could engraft and function. Selawry and Whittington11 transplanted islet grafts to the testis, another immune protected site, and found that such grafts induced euglycemia when the testis was placed within the abdominal cavity (leaving the testes within the scrotum did not seem to allow metabolic control of hyperglycemia). Thus, a number of different strategies seemed to allow successful islet transplantation across alloimmune barriers without the need for chronic immunosuppression in various small animal models, and the prospect that this might be achievable in large animals and then in humans stirred considerable enthusiasm. Yet, 30 years ago, successful islet transplantation in diabetic humans remained elusive. In 1984, Sutherland et al52 published a review of such attempts, along with a review of all whole organ pancreas transplants performed for diabetic patients to that date, an astonishing effort that would now be nearly impossible to duplicate.

CLINICAL ISLET TRANSPLANTATION The Early Years Through the 1980s, the following 3 formidable barriers stood in the way of islet transplantation as a treatment for diabetes in humans: lack of a method to consistently isolate a sufficient number of viable islet from the human pancreas, lack of an effective nontoxic and in particular nondiabetogenic regimen of immunosuppression able to prevent alloimmune and autoimmune damage of the islet graft, and a shortage of a donor source of human pancreas. The first 2 barriers were considered so high as to dwarf the third, but now, the availability of human pancreas is really the limiting factor in clinical human islet © 2015 Wolters Kluwer Health, Inc. All rights reserved.



transplantation just because it is the limiting factor in all organ replacement therapy. Around the 10-year anniversary of Pancreas, the process of islet isolation, as first described in rodents, was used in an upscale fashion to isolate islets from large animal pancreases for research and the human pancreases for research and clinical applications. Although there was some variability from laboratory to laboratory, the process basically employed pancreatic duct injection of a collagenase solution followed by some form of mechanical separation of the tissue, filtration, and density gradient purification. The handling of large amounts of tissue, especially during the collagenase digestion phase, was cumbersome and even “messy” prolonging the amount of time necessary to process the islet tissue and allowing inadvertent contamination with adventitious organisms. Development of a digestion chamber allowed semiautomation of this part of the islet isolation procedure, thereby decreasing direct tissue handling and standardizing perhaps the most inconsistent part of the isolation procedure (other than the collagenase used). Several groups contributed to the design, implementation, and popularization of the digestion chamber,33,53 but the device is often referred to as the Ricordi chamber for Ricordi’s application of the device to porcine and human islet isolation.54,55 Other improvements in islet isolation process resulted from the ability to identify islet tissue by specific staining (dithizone) and therefore quantify islets in a consistent manner.56,57 Heretofore, islets were counted on the basis of morphology or semiquantified by measuring insulin content, the former subjective, the later objective, but imprecise. The use of dithizone to identify islet tissue and quantification of islet equivalents (IEQs, normalizing islet counts based on their size) allowed better comparison of islet isolation outcomes both within the same laboratory but also between islet centers and groups studying isolation methodology.58,59 Consolidation of process methods (isolation, quantification, viability testing) and standardization within the field of islet transplantation are important aspects of achieving regulatory approval for clinical application. This was achieved among islet transplant programs in the recent multicenter phase 3 islet transplantation study.25 Development of the digestion chamber, dithizone staining, advances in viability testing, and improved quantification of islet isolation results advanced the field of islet transplantation considerably, spurring new attempts of clinical islet transplantation for diabetes in the late 1980s and 1990s.60–67 Enthusiasm about the potential of the approach for treatment of diabetes remained high and many predicted that its success was “just around the corner.” It was during this same era that whole organ pancreas transplantation became well established as a successful clinical option for patients with type 1 diabetes.52,68 It has been natural to compare the relatively highly successful but “morbid” pancreas transplant with the less morbid but also considerably less successful islet transplant. It is the author’s opinion that these comparisons have not always benefited the field of replacement therapy for treatment of diabetes. Although the first attempts of islet transplantation in humans were performed in the 1970s,32,69 improvements in islet isolation and a new hope for successful clinical islet transplantation increased islet transplant numbers. By December 31, 1995, a total of 270 islet transplants (at >30 institutions worldwide) had been performed for patients with type 1 diabetes, with an overall insulin-independence rate of 10%.70 Of the 270 insulin-dependent patients who received an islet transplant, 14 achieved insulin independence lasting 1 year or more. The majority demonstrated increased C-peptide production despite remaining diabetic, implying that most recipients had a functional islet graft but had either received an insufficient


mass of islets, their grafts functioned poorly, or both. This observation was important for the next era of islet transplantation.

Modern Era of Clinical Islet Transplantation Strategies in small animal models of islet transplantation seemed to allow islet engraftment and long-term function without the need for chronic immunosuppression. For example, UV radiation, a period of islet culture, use of a small number of islets from multiple donors, or specific means to eliminate or reduce islet contaminating antigen-presenting cells permitted islet graft acceptance in certain rodent islet transplant models.42,43,48,71 Immunoisolation (microencapsulation or macroencapsulation) or use of immunoprivileged sites allowed islet transplantation without the need for immunosuppression in these same small animal models.11,39 However, none of these strategies worked consistently in large animals and the need for immunosuppression in human clinical islet transplant trials was widely accepted. Unfortunately, most immunosuppression strategies used for solid organ transplantation were considered highly diabetogenic because they used corticosteroids. In the 1990s, many solid organ transplant recipients were treated with an induction immunosuppressive agent (usually T-cell–depleting antibody or antibodies) followed by “triple” immunosuppression maintenance comprised of a calcineurin inhibitor (eg, cyclosporine or tacrolimus), a DNA antimetabolite (eg, azathioprine or mycophenolic acid), and steroid. Nearly all clinically useful immunosuppression agents demonstrate either β-cell toxic effects in vitro or diabetogenicity in vivo (perhaps the exception being the antimetabolites), but steroids are generally considered the most diabetogenic.72–76 Recognizing that not all renal transplant programs used steroid as part of the maintenance regimen, Shapiro et al77 designed a steroid-free immunosuppressive protocol for human islet transplantation. Also, recognizing that nearly all clinical islet transplant recipients demonstrated partial function (based on C-peptide data), a clinical trial was designed where multiple pancreas donors could be used.77 In 2000, Shapiro et al77 published a series of 7 type 1 diabetic patients independent of insulin after islet transplantation. The steroid-free immunosuppressive protocol consisting of daclizumab (an IL-2 receptor antagonist), sirolimus, and low-dose tacrolimus used in these patients came to be known as the “Edmonton protocol.” The success of the protocol seemed related both to the steroid-free immunosuppression regimen and use of multiple donors (up to 3 donors) to provide a large islet mass. In a follow-up paper from the University of Alberta group, Ryan et al78 reported that 11 of 12 patients became insulin independent after receiving a minimum of 9000 IEQ per kilogram of recipient body weight. After a median of 10 months, 4 patients had normal glucose tolerance, 5 patients had impaired tolerance, and 3 patients had diabetes.78 The success of the Edmonton group resulted in renewed efforts by experienced islet transplant programs and the development of new islet transplant centers. An international multicenter trial enrolled 36 subjects with type 1 diabetes who underwent islet transplantation using the Edmonton protocol. Twenty-one (58%) became insulin independent at some point after transplant; 5 remained insulin independent at 2 years.79 The results were a confirmation of the Edmonton protocol and represented a considerable advance for the field of islet transplantation, but it was clear that islet transplantation had not surmounted all barriers.80 Infusion of a larger than expected islet mass was required for achievement of insulin independence, insulin independence was not always achieved, and function declined or was lost in the longer term in most recipients.

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A common reason for loss of function after allogeneic transplant is immunological: damage of the graft due to rejection. Detection of donor-specific antibody and cellular immune responses against mismatched antigens infers that immune-mediated injury of human islet grafts does occur.81–84 However, definitive diagnosis of rejection, acute or chronic, is typically made by biopsy. For whole organ transplant, biopsy is an established procedure and is relatively straightforward, but for islet grafts, tissue diagnosis of rejection is less common. Intrahepatic islet grafts have been identified by hepatic biopsy,85 but a concerted effort to perform a biopsy on the liver to sample intrahepatic islet grafts in the setting of declining graft function (another relatively difficult endpoint to measure) has not been done clinically. Thus, the reason(s) for long-term islet graft failure remains speculative. Given that immune mechanisms likely play a role in decline of islet graft function in humans, more recent clinical islet transplant trials have used alternative immunosuppressive strategies. Using T-cell–depleting induction immunosuppression, tumor necrosis factor or TNF inhibition, and an initial maintenance immunosuppression regimen of cyclosporin and everolimus, Bellin et al86 at the University of Minnesota achieved long-term insulin independence in 4 of 6 type 1 diabetic patients who received islet transplants. In another trial, a steroid-free and calcineurin-free immunosuppression regimen including efalizumab was associated with insulin independence in 8 of 8 type 1 diabetic islet transplant recipients at the University of California, San Francisco.87 Immunosuppressive modifications of the Edmonton protocol seem to be making iterative improvements in clinical human islet transplantation outcomes. For the past 30 years, islet transplantation has developed into a conceptually valid replacement therapy for some patients with severe labile type 1 diabetes. A review of 677 islet transplants alone or islet after kidney transplants reported to the Collaborative Islet Transplant Registry between 1999 and 2010 shows greater than 40% 3-year insulin independence in a 2007 to 2010 cohort of 208 recipients.88 Recently, a phase 3 multicenter study of islet transplantation for prevention of hypoglycemia in severe labile diabetic patients completed enrollment. Hering et al25 presented preliminary results from this study at the 2014 meeting of the American Diabetes Association. Approximately 55% of patients were insulin independent at 1 year after transplant, but a large majority had improved blood glucose control and fewer episodes of hypoglycemia. The results from this study might be used for application for Food & Drugs Administration approval for clinical islet transplantation. Since the first issue of Pancreas, the field of islet transplantation has moved from proof of concept in small animal models to clinical trials in patients with type 1 diabetes. A large amount of experimental work and clinical translation has taken place for more than 3 decades. However, islet transplantation, because it currently exists, has not yet achieved the potential of β-cell replacement therapy. Long-term results of islet transplantation lag behind that of whole organ pancreas transplantation, and immunosuppression remains necessary despite early hopes, based on small animal models, that tolerance to islet grafts might be achieved. Although human islet transplantation is now feasible, broader application of this β-cell replacement therapy will require improvement in metabolic function, avoidance of the immune system, and means to address the shortage of organs for donation. Pagliuca et al89 recently described the ability to generate large numbers of functional human β cells from pluripotent stem cells. Although this approach may meet the need for an inexhaustible source of donor tissue, presumably host immune

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barriers will remain a challenge. Encapsulation, as described in the following section, is a tool that may address a number of these issues.

MICROENCAPSULATION OF ISLETS FOR TRANSPLANTATION Rationale for Islet Microencapsulation As obvious from the preceding sections, it had become clear that the use of immunosuppressive drugs to prevent transplant rejection was toxic to both islets and transplant recipients. In addition, it had always been a concern that if even we could get alloislet transplantation to work in patients, it would be difficult to use it routinely to treat type 1 diabetic patients because of the severe shortage of human pancreas. Therefore, an approach to encapsulate islet cells emerged as an immunoisolation strategy to overcome the major barriers to islet transplantation, namely, the need for immunosuppression and the shortage of human pancreas. Two forms of encapsulation have been examined. In macroencapsulation, multiple islets are encapsulated in 1 or several large capsules whereas microencapsulation entails the encapsulation of 1 or 2 islets in 1 semipermeable microcapsule typically measuring less than 1 mm in diameter.90–95 The major drawback of macrocapsules is the relative low surface-to-volume ratio, which interferes with optimal diffusion of nutrients and oxygen. For adequate supply of nutrients and oxygen, the islet density in the macrocapsules is kept quite low (usually 5%–10% volume). This makes the macrocapsules rather impractical because large devices have to be implanted to provide sufficient masses of islets and these devices cannot be implanted at conventional transplantation sites.96 Low surface-to-volume ratio also interferes with glucose regulation because of slow exchange of glucose and insulin.94 Consequently, more attention has been paid to microencapsulation of islets, which is currently the preferred approach to the immunoisolation of islets for transplantation.90,92–95,97 This discussion will therefore focus on microencapsulation.

Nuts and Bolts of the Microencapsulation Technology The operational principle of this technology is illustrated in Figure 2, and a few years before the birth of the Pancreas, Lim and Sun98 had first reported that successful single implantation of microencapsulated islets into rats with streptozotocin-induced diabetes corrected the diabetic state for 2 to 3 weeks. The microencapsulated islets remained morphologically and functionally intact throughout long-term culture studies lasting for 15 weeks.98 Figure 3 represents a sample of microencapsulated islets made in our laboratory at the Wake Forest Institute of Regenerative Medicine at the Wake Forest School of Medicine in WinstonSalem, NC. Alginate is the most studied hydrogel for islet microencapsulation, because it provides some major advantages more than other encapsulation materials. Alginate molecules are linear block copolymers of β-D-mannuronic (M) and α-L-guluronic acids (G), which, under physiological conditions, readily form a gel in the presence of divalent ions such as Ca2+ and Ba2+,90,92–95,97,98 in contrast to other polymeric materials, such as agarose, polyethylene glycol, nitrocellulose acetate, 2-hydroxyethyl methacrylate (HEMA), acrylonitrile, polyacrylonitrile and polyvinylchloride copolymer, and sodium methallylsulfonate, which have also been studied for islet microencapsulation.90,91,93–95,99,100 It has been shown that divalent ions cross-link not only G blocks but also blocks of alternating M and G (M-G blocks).101 Calcium has been the main cation used for gelling, because barium is known to be © 2015 Wolters Kluwer Health, Inc. All rights reserved.




FIGURE 2. Illustration of the principle of microencapsulation technology. Reprinted with kind permission from Opara et al.92

toxic and concerns have been raised about patients’ safety if it is used in cross-linking microcapsules for islet transplantation. As noted earlier, alginate is a unique polymeric material that allows islet encapsulation under physiological conditions. The encapsulation can be done at room or body temperature, at physiological pH, and in isotonic solutions. Islets have been shown to more readily and adequately survive when enclosed in alginate microcapsules.102 It does not interfere with the cellular function of the islets.91,92 It has been shown that alginate capsules provide a microenvironment facilitating functional survival of islets probably because the 3-dimensional matrix provides mechanical support for the islets and prevents clumping and fusion of the free islets, thus preserving islet structure. Alginate-based microcapsules have been shown to be stable for years in both animals and human.91 Alginate has been found to be highly biocompatible because alginate molecules are negatively charged and the attachment of immune cells to the alginate microcapsule is limited owing to the negative charges on the cell surface.

Permselective Coating of Alginate Microcapsules for Immunoisolation of Islets Uncoated nonpermselective alginate microbeads have been reported to have high permeability for molecules with molecular sizes greater than 600 kD. Uptake studies with IgG (150 kD) and thyroglobulin (669 kD) have suggested that these molecules are able to get into these uncoated microbeads. Similarly, uncoated alginate microbeads implanted in the peritoneum were positive for both IgG and C3 component after only 1 week.103 Therefore, molecules with a range of sizes from macrophages and T cells to smaller cytokine molecules such as IL-1β, TNF-α, and IFN-γ can easily penetrate into the alginate microcapsules and cause damage or destruction of the encapsulated islets.104 To provide immunoisolation for the microcapsules, it is essential to apply a permeability barrier between the encapsulated islets and host immune system. Coating the alginate microcapsules with a polyamino acid layer, followed by an additional outer coating with alginate, typically creates this barrier. The positively charged polyamino acid molecules will readily bind to the negatively charged alginate molecules forming a complex membrane,105 which significantly reduces the pore size of the microcapsule and prevents immune cells from entering the microcapsule. To prevent interactions of nonbound polyamines to host tissue, a thin second layer of alginate is added.91–93 The polyamino barrier acts as a shell, providing mechanical stability to the microcapsule, allowing for the liquefaction of the inner alginate.106 The

thickness of this barrier can be varied through incubation time and concentration.91 The most routinely used permselective biomaterial is poly-L-lysine, which was the first material used to create this barrier;98 however, more recent research has shown that poly-L-ornithine coating provides more mechanical support to the microcapsules with markedly reduced immune response.107,108 It had been previously reported that conventional immunoisolatory coating of microcapsules may not prevent permeability of microcapsules by most cytokines and chemokines.109,110 However, de Vos and Marchetti97 have indicated that cytokines released by the host immune system might not represent an insurmountable shortcoming for clinical application of the immunoisolated islet transplantation. In particular, they found evidence that cytokines might not interfere with islet function after xenoislet transplantation in humans because they observed that after exposure to a combination of human cytotoxic cytokines, a marked decrease in functional survival and a high proportion of apoptotic cells could be seen in human islets but not bovine islets.111 These data suggest that it is likely that even when microcapsules may be permeable to cytokines, the function and survival of xenogeneic islets will be less vulnerable compared with encapsulated human islets.

FIGURE 3. Microencapsulated islets in alginate microcapsules. Scale = 100 μm.




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Current Challenges and Approaches to Solving Them Because the introduction of the microencapsulation technology in islet transplantation research significant attempts have been made toward clinical translation as reviewed in the Clinical Applications of Microencapsulated Islets.98 However, there are certain challenges that still prevent this promising technology from full realization of its full clinical potential, as previously discussed.90,92–95 Some of the pressing challenges facing the microencapsulated islet transplantation include the following: internal oxygen and nutrients transfer limitation to encapsulated cells that causes severe hypoxia and massive death of β cells, reducing the total transplant volume, reduction of inflammatory response after transplant of the encapsulated cells including consequence of incomplete encapsulation of all the cells, need for transplantation at retrievable and vascularized sites for sustained graft function and posttransplant assessment, and scale-up devices for microencapsulation.112 In various studies for the last decade, researchers across the globe are examining different strategies to address these barriers to clinical application of microencapsulated islet transplantation, which we will now discuss in more details along with recent research strategies that have been presented toward overcoming them.

Internal Oxygen and Nutrient Transfer Limitation to Encapsulated Cells Cell survival in microcapsules is critically dependent on the supply of nutrients and oxygen. Hypoxia remains a major concern with encapsulation particularly in encapsulated islets transplanted in the peritoneal cavity that lacks an organized vasculature.92,113 Oxygen diffusion is limited to a distance of ∼100 μm in tissue,114 which is exceeded when islets with an average diameter less than 200 μm are enclosed in standard microcapsules with average diameters greater than 700 μm, as seen in most published studies. As a result, most studies with encapsulated islets have used extraordinarily high doses of these cells to achieve variable effects on blood glucose levels in large animals and human subjects.92 It is necessary to keep in mind that different polymeric materials such as agarose and alginate used for microencapsulation have different permeabilities to oxygen,115 and it has been suggested that different oxygen delivery systems such as microparticulate and nanoparticulate oxygen generators and perfluorocarbon may be included in an encapsulation matrix as a useful strategy for overcoming oxygen diffusion limitations and enhance cell viability and functionality long term even in large devices with diameters greater than 1 mm.113,116 It has also been suggested that the survival and function of encapsulated islets may be improved with stimulation of local neovascularization with growth factors,92 albeit this may depend on the transplantation site.117 The recent report by Ludwig et al118 of a new implantable device containing microencapsulated islets that not only provides the donor islets immunological protection from the host immune systems but also provides them with controlled and adequate oxygen supply is certainly a step in the right direction.

Search for an Optimal Transplantation Site Determining the optimal site for systemic drug delivery is a matter of intense research. Although, because of technical ease of accessibility and its huge capacity to accommodate a large transplant volume, the peritoneal cavity has been used in most studies with encapsulated islets, the optimal site of encapsulated islet transplantation is yet to be determined. There seems to be a consensus that vascularized sites would be better than the peritoneal cavity because of the high oxygen requirements of islets.

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Therefore, investigators have been looking at other transplant sites such as the subcutaneous space, the kidney capsule, and the omentum. Several factors are to be taken into consideration including biocompatibility, mechanical resistance, ability to revascularize quickly, and ability to be retrieved after prolonged periods of transplantation for further studies. For example, capsules implanted intraperitoneally showed decreased viability, insulin secretion rates, and higher immune response as compared with capsules implanted subcutaneously or under the kidney capsule.119 These observations are consistent with a previous suggestion that investigators should focus on finding or creating a transplantation site that, more than the unmodified peritoneal cavity, permits for close contact between the blood and the encapsulated islet tissue.120 Our group has recently proposed that transplantation of microencapsulated islets in the omentum via an omentum pouch should be adequate to meet the oxygen needs of islets and easy retrieval of the encapsulated islets for posttransplant analyses.92,117

Reducing the Total Transplant Volume Reducing the total volume of transplanted cells is another major concern for wide implementation of the microencapsulated islet technology. Large capsules limit the transfer of oxygen and other nutrients to the encapsulated cells and add to unnecessary increase of the transplant volume. This issue is also related to the manufacturing process of encapsulated islets because a major challenge facing the technology is to keep the proportion of empty microcapsules present with those containing cells to less than 5% to avoid an unnecessary increase in the transplant volume. One way to reduce this dead volume is to have an effective sorting technique to sort the empty capsules from the microcapsule preparation. Ideally, the sorting would have to be done because the microcapsules are generated to prevent the tedious and timeconsuming sorting process after the empty capsules are mixed with encapsulated islets.112 In recent years, new techniques, such as conformal coating and islet enclosure, molecular entrapment, and nanoencapsulation of islets have been developed.112 With the use of these techniques, the size and total volume of microencapsulated islets for transplantation can be greatly reduced. Although conformal coatings on cell aggregates offer a significant decrease in void volume relative to conventional microcapsules, immunoisolation may, in principle, be accomplished using coatings or membranes of submicron, or nano scale, thickness.121

Tracking of Microencapsulated Islet Transplants Currently, the only way to assess the functional state of microcapsules once they are transplanted is through invasive recovery surgery. There is a growing interest to improve the ability to monitor the implanted cell-loaded devices. An interesting approach to address this need that has been recently proposed is to use alginate-based radiopaque microcapsules containing either barium sulfate or bismuth sulfate, which could be monitored by x-ray.122 Another study has also reported successful application of bioluminescence for real-time tracking of microencapsulated recombinant insulin-producing cells.123 It remains to be determined whether any of these emerging techniques would find relevance in clinical transplantation of microencapsulated islets.

Complete Encapsulation of Cells Complete encapsulation of cells is of considerable importance to prevent an immune response from the host system. This is another area of significant challenge in the manufacturing of © 2015 Wolters Kluwer Health, Inc. All rights reserved.



microencapsulated islets because the ideal microcapsule size should be 400 to 500 μm in diameter. It has been reported that decreasing the size of alginate microcapsules from 800 to 500 μm is associated with a ∼4-fold increase in the percentage of incompletely encapsulated islets.124 Host response to even 2% to 10% of encapsulated islets has been shown to result in the destruction of 40% of the graft.120,125 Therefore, complete encapsulation of all transplanted cells remains an important prerequisite for successful immunoisolation.

Scale-up Devices for Microencapsulation One of the biggest challenges facing the field of cell microencapsulation is scaling up of the manufacturing process. The 2 most widely used devices for microencapsulation are the air-syringe pump droplet generator and the electrostatic bead generator.126–128 Each of these devices is fitted with a single needle through which droplets of cells suspended in alginate solution are produced and cross-linked into spherical microbeads. A major drawback in the design of these instruments is that they are incapable of producing sufficient numbers of microcapsules in a short-time period to permit mass production of encapsulated and viable cells for transplantation in large animals and humans.92,129 A prolonged process of encapsulation of cells adversely affects the viability of the cells. A multi-needle approach to producing more than 1 encapsulated cell at a time as a scale-up of the process has been described with 4 needles.129 Although this scale-up is a step forward in accelerating the production of encapsulated cells, production rates at several orders of magnitude higher are required to meaningfully produce sufficient quantities of encapsulated and viable cells to serve millions of patients requiring cell transplantation. For instance, for transplantation in human subjects, it has been estimated that for the 1 million islets needed for transplantation in a diabetic human subject, approximately 100 hours would be required to complete the encapsulation of this number of islets, assuming 1 islet per microcapsule. In practice, it has actually been estimated that the duration of the process would be closer to 200 hours because of the additional steps involved in the encapsulation procedure, after the generation of the initial cell containing alginate microspheres.129 As exhaustively reviewed recently,112 studies have reported some promising approaches to scaling up of the production rates of microencapsulated islets. These include vibrational technology with multi nozzle design for scaling up the production of alginate beads, shear flow–driven methods and electrospraying techniques, and others such as the recently introduced microfluidic approach.130 However, there is currently no commercially available microencapsulation device that satisfactorily meets the optimum requirements for islet microencapsulation such as acceptable speed to microencapsulate islets for the millions of available patients with type 1 diabetes and reproducibility in perfections of spherical shape and size for optimum function of the encapsulated cells. Extensive and routine clinical use of microencapsulated islets to treat diabetic patients is anxiously waiting for the successful development and availability of a reliable mass production device for manufacturing microencapsulated islets for transplantation.

Clinical Applications of Microencapsulated Islets Despite the overwhelming obstacles to clinical translation of the microencapsulated islet technology, there has been a string of clinical trials with encapsulated islets starting with the first report by Calafiore131 in 1992 after his group had described a special coaxial vascular prosthesis in which microencapsulated islets could be placed with direct access to blood flow.132 This group


subsequently used this strategy to explore vascular grafting of microencapsulated islets in dogs and 2 human patients, one with type 1 diabetes and the other with type 2 diabetes managed with insulin.131 After undergoing transplantation, the patient with type 2 diabetes showed a steady decrease in exogenous insulin requirement, and serum C-peptide rose progressively both at baseline and with stimulation and peaked at day 18 posttransplantation when exogenous insulin was temporarily withdrawn. The efficacy of the transplant as judged by a 5-fold decrease in daily exogenous insulin administration was sustained for a 7-month period. The patient with type 1 diabetes showed early appearance of C-peptide, which was not detectable before transplantation. The C-peptide levels increased during 60 days of clinical follow-up and were associated with improvement in blood glucose control. This report thus demonstrated that immunoisolation of islets in highly biocompatible and selective permeable microcapsules may effectively immunoprotect islet graft tissue from the host’s immune response. Shortly afterwards, Soon-Shiong et al133 reported insulin independence in a type 1 diabetic patient after encapsulated islet transplantation. Microencapsulated human islets were injected intraperitoneally in a diabetic patient with functioning kidney graft, and this resulted in insulin independence with tight glycemic control for 9 months after the procedure. Then came the first report of encapsulated pig islet xenotransplantation in a type 1 diabetic patient from a group led by Elliott et al134 in New Zealand. In their protocol, the encapsulation process involved the preparation of alginate-poly-L-lysine-alginate microcapsules similar to those described by Lim and Sun98 and then enclosing them in a larger similar capsule. A 41-year-old white man with type 1 diabetes who was free of any clinically detectable diabetic complications but had poor glycemic control was approved to receive the xenotransplantation. The dose implanted was 15,000 IEQs/kg body weight and a total of 1.3 million IEQs of the alginate encapsulated neonatal porcine islets were placed intraperitoneally. The average insulin dose after transplantation was reduced by up to 30% for the next 14 months after which the daily insulin used returned to the pretransplant level. However, at laparoscopy performed 9.5 years after transplantation, abundant nodules were seen throughout the peritoneum, and biopsies of the nodules showed opacified capsules containing cell clusters that stained as live cells under fluorescence microscopy. The retrieved capsules produced a small amount of insulin in vitro when placed in high glucose concentrations, thus indicating the presence of live encapsulated porcine islets after nearly 10 years of transplantation in a human diabetic patient.134 In 2006, the Calafiore et al135 again reported the first 2 cases of microencapsulated islet allografts in the peritoneal cavity of immune-competent patients with type 1 diabetes in their phase 1 pilot study with microencapsulated islets. Both patients showed amelioration of their mean daily blood glucose levels and a progressive decline in exogenous insulin requirements. At 60 days posttransplantation, an oral glucose tolerance test in the patients showed a biphasic C-peptide response compatible with the presence of functional β cells. About the same time, another group led by Tuch et al136 in Sydney Australia started a safety and viability test of microencapsulated human islets in diabetic human patients. This group studied 4 type 1 diabetic patients with no detectable C-peptide who received an intraperitoneal infusion of islets encapsulated in alginate microcapsules cross-linked with barium. They reported that C-peptide was detected 1-day posttransplantation and that blood glucose levels and insulin requirements decreased, but C-peptide was undetectable by 1 to 4 weeks. However, in a multi-islet transplant recipient, C-peptide was detected at 6 weeks after the third infusion and remained




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detectable for 2.5 years. In 2011, the Calafiore group extended their clinical trial to 4 nonimmunosuppressed cases.137 The patients received intraperitoneal transplantation of microencapsulated human islets. All patients turned positive for serum C-peptide detection both under basal and stimulated conditions throughout 3 years of posttransplant follow-up. Daily mean blood glucose and Hba1c levels significantly improved after transplantation with insulin dose decreasing in all cases and even being discontinued transiently in 1 patient. More importantly, the grafts did not elicit any immune response even in the cases of multiple transplantations. Driven by their initial observations the New Zealand group’s commercial arm, the Living Cell Technologies (LCT Global) commenced formal clinical trials with their encapsulated neonatal islet xenotransplant program beginning with phase 1/2a clinical trials in Russia in 2009. According to the information available from the LCT website,138 in the Russia trial, a total of 8 patients received the microencapsulated pig islet implants of varying doses. Some patients received multiple doses. Data analysis confirmed that the trial successfully met its endpoints of demonstrating safety and tolerability. In addition, the trial showed proof of principle of efficacy in humans with type 1 diabetes. Six of the 8 patients on the trial demonstrated improvements in blood glucose control as reflected by reduced HbA1c levels and reduced dose of daily insulin injections. Two patients discontinued insulin injections entirely for up to 32 weeks. The LCT clinical trials have now advanced to phase 2a safety and efficacy studies in New Zealand and Argentina. The on-going trial in Argentina involves 8 patients split into 2 groups of four. Group 1 received two 5000 IEQ/kg doses of microencapsulated porcine islets (IEQs per kilogram of body weight). Group 2 received two 10,000 IEQ/kg doses. In both groups, the second dose was implanted 12 weeks after the first. The most significant observations from the LCT clinical trial in Argentina are reduction of average insulin dose by 20%, a reduction of HbA1c levels from a pretransplant average of 8.6% to 6.7% at 12 weeks after the second transplant, and up to 70% reduction in hypoglycemia unawareness. The phase 2a clinical trial in New Zealand has now been completed. It involved 16 patients with type 1 diabetes treated at Middlemore Hospital in Auckland. The patients were treated with different levels of the following islet cell doses: 5000, 10,000, 15,000, and 20,000 IEQ/kg body weight. At 52 weeks, HbA1C levels were reduced in the 5000, 10,000, and 20,000 IEQ/kg treatment groups, compared with baseline. A statistically significant reduction in the number of unaware hypoglycemic episodes was observed in both 5000 and 10,000 IEQ/kg groups. A reduction in insulin use was evident in all treatment groups, with marked mean reductions in the 5000 and 10,000 IEQ/kg groups. The islet cell implants were well tolerated in all patients. Quality of life questionnaires revealed a positive impact of treatment. In 2012, Ludwig et al118 described a macrochamber specially engineered for islet transplantation. The subcutaneous implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 months.118 In the Ludwig device, encapsulated islets are maintained within an alginate slab configuration adjacent to an oxygen-permeable membrane, which creates a sufficient immune barrier and allows for adequate oxygen supply to the islet graft. The investigators achieved enhanced function of encapsulated islets by pretreating them with growth hormone releasing hormone analogs. Sufficient graft function depended on oxygen supply. This device popularly referred to as the bioartificial pancreas (BAP)—the “β Air”139—seems to provide a strategy capable of restoring and maintaining long-term

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euglycemia in diabetic models and is currently undergoing clinical trial in the United Kingdom and perhaps elsewhere in Europe. At the present time, although microcapsules using alginate or other polymeric hydrogels to encapsulate individual islets have shown great promise in hundreds of rodent studies, the technology has had limited success in large animal and human trials, as recently reported.95 However, investigators have remained actively engaged in exploring the full potential of the microencapsulated islet technology because of its great appeal as a strategy to avoid the immunosuppression of transplant recipients, the expansion of the islet donor pool, and the technical ease of transplantation. Interestingly, newer approaches to improving the viability and function of microencapsulated islets are emerging. One of these approaches with great appeal involves the co-encapsulation of islets with extracellular matrix proteins and mesenchymal stromal cells.140,141 The rationale in this elegant approach is to re-establish the natural islet microenvironment lost during cell isolation and for the mesenchymal stromal cells to provide immunomodulatory properties and/or enhance islet survival and function. Another group has also shown that co-encapsulation of bioengineered IGF-II–producing cells and islets promotes islet cell survival.142 Although these newer approaches remain to be put to the clinical test, they provide sufficient ground for optimism such that one can speculate with good reason that it is only a matter of time before the microencapsulated islet technology achieves full clinical translation.

SUMMARY AND CONCLUSIONS The first attempts at β-cell replacement in humans, pancreas and islet transplantation, were performed in the 1960s and 1970s. Although pancreas transplantation has been an accepted treatment for severe labile diabetes for many years,143 allogeneic islet transplantation remains experimental. Approved clinical islet transplantation is limited to autologous islet transplantation to prevent diabetes in patients who undergo extensive pancreatectomy for benign pancreatic diseases.144 Current investigations in the field of islet transplantation focus on means to improve islet function after transplantation. Improving islet viability during isolation, exploring ways to increase engraftment, and protection from the host immune system are some of the goals of these investigative efforts. Encapsulation, as described in the preceding section, is a tool that can be modified to address many of these goals. In addition, if regenerative medicine achieves the means to provide “new” islets or infectious and ethical problems related to xenografts might prove acceptable, encapsulation technology might be employed to provide the appropriate environment for engraftment of an islet transplant that faces any type of immune barrier. To improve the outcome of islet transplantation, 1 area of significant importance is coming up with a reliable islet isolation procedure that will consistently result in functionally viable islets before transplantation and in the immediate posttransplant period. Specifically, the current isolation technique of collagenase enzyme-based digestion of pancreatic tissue, which perturbs the tissue matrix and adhesion molecules, thereby impairing the function of isolated islets,145 needs to be revisited. The emergence of nonenzyme-based procedures for islet isolation146,147 is therefore quite timely, and these techniques need to be optimized for human islet isolation. These procedures would remove collagenaseinduced damage to islets, endotoxin contaminations, and the high cost and laboriousness of islet isolation. In particular, the selective osmotic shock procedure would enable islet isolation regardless of the texture of the pancreas, thus ensuring reproducibility. Above all, these nonenzymatic procedures may eliminate the need for the expensive equipment required for islet purification. © 2015 Wolters Kluwer Health, Inc. All rights reserved.



Furthermore, it is necessary to keep in mind that the current procedures for islet isolation do not take into consideration the fact that islets are very highly dependent on oxygen for function, which therefore makes it mandatory for us to come up with better techniques for oxygen supplementation during the islet isolation process. New strategies are emerging to address this important issue, which is also important when dealing with encapsulated islets.113 The high oxygen dependence of islets also needs to be taken into consideration in determining the optimal site of transplantation, which ideally needs to be vascularized for ease of oxygen and nutrient delivery to the islets. With improved islet isolation techniques and determination of the best site of engraftment as well as improved encapsulation techniques, it is certainly possible that islet transplantation could someday achieve routine clinical application. REFERENCES 1. Bliss M. The Discovery of Insulin. Chicago: The University of Chicago Press; 1982. 2. The Diabetes Control and Complications Trial Research Group. The absence of a glycemic threshold for the development of long-term complications: the perspective of the Diabetes Control and Complications Trial. Diabetes. 1996;45:1289–1298. 3. Hampton T. Fully automated artificial pancreas finally within reach. JAMA. 2014;311:2260–2261. 4. The Diabetes Control and Complications Trial Research Group. Hypoglycemia in the Diabetes Control and Complications Trial. Diabetes. 1997;46:271–286. 5. Kelly WD, Lillehei RC, Merkel FK, et al. Allotransplantation of the pancreas and duodenum along with the kidney in diabetic nephropathy. Surgery. 1967;61:827–837. 6. SRTR. In: Services USDoHaH, ed. Annual Report of the U.S. Organ Procurement and Transplantation Network and the Scientific Registry of Transplant Recipients: Transplant Data 2002-2011. Rockville, MD: Health Resources and Services Administration, Healthcare Systems Bureau, Division of Transplantation; 2011. 7. Sutherland DE, Goetz FC, Rynasiewicz JJ, et al. Segmental pancreas transplantation from living related and cadaver donors: a clinical experience. Surgery. 1981;90:159–169. 8. Farney AC, Rogers J, Stratta RJ. Pancreas graft thrombosis: causes, prevention, diagnosis, and intervention. Curr Opin Organ Transplant. 2012;17:87–92. 9. Sutherland DE, Goetz FC, Sibley RK. Recurrence of disease in pancreas transplants. Diabetes. 1989;38(suppl 1):85–87.


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