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A biologic resorbable scaffold for tissue engineering of the endocrine pancreas: Clinical experience of islet transplantation on the omentum
C H A P T E R
19 A biologic resorbable scaffold for tissue engineering of the endocrine pancreas: Clinical experience of islet transplantation on the omentum David A. Baidal⁎,†, Marco Infante⁎,‡, Virginia Fuenmayor⁎, Ana M. Alvarez⁎, Nathalia Padilla⁎, Dora M. Berman⁎,‡, Antonello Pileggi¶, Elina Linetsky⁎,§, Gaetano Ciancio§, Camillo Ricordi⁎,§,ǁ, Rodolfo Alejandro⁎,† ⁎
Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States †Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, United States ‡Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy §Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States ¶Division of Physiology and Pathological Sciences, Center for Scientific Review, National Institutes of Health, Bethesda, MD, United States ǁcGMP Facility, Cell Transplant Center, Division of Cellular Transplant, Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States
O U T L I N E Introduction: The intrahepatic site for islet transplantation
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Extrahepatic sites for islet transplantation
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The greater omentum: A novel site for islet transplantation
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Introduction: The intrahepatic site for islet transplantation Islet transplantation (ITx) is a cellular therapy that has demonstrated efficacy in improving glycemic control and abolishing hypoglycemia in patients affected by type 1 diabetes (T1D) complicated by hypoglycemia unawareness and episodes of severe hypoglycemia.1–3 However, it has been a long road prior to achieving the success rates that are currently observed with ITx. Prior Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00019-1
Preclinical experience Clinical experience
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Conclusions
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References
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to 2000, ITx efforts throughout centers worldwide were met with very limited function evidenced by <10% of patients achieving insulin independence by 1 year posttransplantation.4 It was not until the introduction of the Edmonton protocol in 2000 that intrahepatic ITx really surfaced as a successful therapeutic modality for adult patients affected by T1D.5 Shapiro et al. reported insulin independence at 1 year on seven consecutive subjects, a breakthrough for the field at the time.5 The key factors associated with the success of this novel transplantation
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© 2020 Elsevier Inc. All rights reserved.
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protocol included the intraportal infusion of a cumulative islet mass of ≥10,000 islet equivalents (IEQ) per kg of body weight obtained from at least two islet infusions, the avoidance of glucocorticoids, induction immunosuppression with IL-2 blockade, and maintenance immunosuppression with the combination of an mTOR inhibitor (sirolimus) and an FK-506 inhibitor (tacrolimus). However, a particular limitation of this protocol was the need to transplant the islets within 4 h from isolation which precluded a more thorough assessment of the islet preparation prior to release for transplantation. The Miami group showed similar success rates using an “islets in culture” protocol compared with the initial Edmonton experience.6 Culturing of islets allowed for additional assessment of the islet product prior to transplantation and for better determination whether the islet product was suitable for clinical transplantation. In 2012, the use of potent induction immunotherapy with thymoglobulin combined with TNF-α blockade demonstrated improved outcomes at 5 years, similar to results observed in pancreas transplantation.7 Despite these improvements in immunosuppressive and antiinflammatory strategies, long-term data of intrahepatic islet allografts show a progressive decline in beta-cell function.8 Several factors may contribute to this loss, including allograft rejection, recurrence of autoimmunity, and metabolic exhaustion.9, 10 In addition, the use of an intravascular site poses several challenges. Intraportal islet infusion is associated with an increase in portal vein pressure that is proportional to the volume and number of the transplanted islets, thus limiting the total islet mass that can be transplanted.11, 12 Other limitations of the intraportal infusion include the potential risk of bleeding and thrombosis.12 In addition, an instant blood-mediated inflammatory reaction (IBMIR) is triggered once islets come into contact with blood.13 The IBMIR results in the activation of the coagulation and complement cascade, leading to formation of clots around the islets which contribute to a significant loss of islet integrity and function.14 Moreover, intrahepatic islet infusion leads to thrombosis and liver ischemia due to islet entrapment in the liver sinusoids, thus resulting in the activation of sinusoidal endothelial cells and Kupffer cells with subsequent islet cell damage and dysfunction.15, 16 In animal models, all the aforementioned ischemia-related events have been shown to result in the loss of approximately 60% of intrahepatic islets due to necrosis and apoptosis observed during the first days posttransplantation.14, 17 The direct exposure of the islet graft to immunosuppressive drugs, nutrients, gut hormones, and metabolites directly from the portal circulation is another important aspect to consider as this may contribute to graft dysfunction.10, 18 As orally ingested immunosuppressive drugs enter the portal circulation, they come into contact with the transplanted islets before undergoing first-pass metabolism.
This leads to the intrahepatic islets being exposed to immunosuppressive drug levels that are higher compared to the systemic circulation, which in turn may result in enhancing drug-related toxic effects on islet function.18, 19 Lastly, revascularization of intraportally transplanted islets takes approximately 7–14 days during which time oxygenation—which is provided by both the portal system and diffusion from the hepatic artery circulation— remains suboptimal, compromising engraftment and aggravating beta cell loss.20, 21 In conclusion, although the intrahepatic site is the only clinical site that has shown islet allograft engraftment with achievement of insulin-independence and long-term graft function, it remains far from being an ideal transplantation site.
Extrahepatic sites for islet transplantation In light of the aforementioned limitations, the identification of an “extrahepatic” site has thus been pursued in the islet transplant field to improve graft engraftment and survival. The optimal characteristics for an ITx site are as follows: • ability to accommodate an adequate islet mass • accessibility by minimally invasive transplant procedures • avoidance or minimization of immune and inflammatory responses • adequate arterial vascular network for nutrient and oxygen delivery • physiologic venous drainage through the portal system • allowing for noninvasive longitudinal morphological and functional monitoring of the islets posttransplants • accessibility for graft biopsy Several preclinical studies have investigated a series of extrahepatic sites for ITx, including pancreas, gastric submucosa, genitourinary tract (i.e., subcapsular renal site, bladder submucosa, and testis), skeletal muscle, subcutaneous space, peritoneal cavity, omentum, spleen, bone marrow, anterior chamber of the eye, thymus, bone marrow, and central nervous system.10, 22 A few of them have been translated to the clinical arena, but have been met with minimal success with the exception of the omentum.
The greater omentum: A novel site for islet transplantation The greater omentum is a highly vascularized structure located in the peritoneal cavity and composed of connective, adipose, and vascular cells, along with specialized immune cells. It has long been considered as an
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The greater omentum: A novel site for islet transplantation
appealing site for ITx as it offers a large and highly vascularized surface area (which may facilitate diffusion of nutrients and oxygen to the graft early after transplantation), exclusive portal drainage, and easy accessibility by minimally invasive procedures.23 Moreover, it displays a remarkable angiogenic potential in response to mechanical and ischemic injury, which may favor islet engraftment and survival.24
Preclinical experience The safety and efficacy of islet allo- and autotransplantation in an omental pouch have been previously demonstrated in preclinical models. Studies in dogs demonstrated that islets infused into an omental pouch restored normoglycemia in pancreatectomized recipients.25, 26 Similarly, Kin et al. found that syngeneic islets transplanted into an omental pouch restored normoglycemia in streptozotocin-induced diabetic rats and nonobese diabetic (NOD) mice.27, 28 Hefty et al. investigated the efficacy of a novel surgical technique for intraomental ITx in dogs.29 The islet pellet was resuspended in phosphate-buffered saline (PBS) containing vascular endothelial growth factor (VEGF). Autologous plasma was then mixed with the islet/VEGF suspension and the resulting coagulum placed onto the omental ventral surface of recipient dogs that previously underwent partial or total pancreatectomy. Then, the leading edge of the greater omentum was rolled up in order to secure the transplanted islets in position and maximize the contact area of the thin islet layer with the two omental surfaces (for nutrient diffusion and subsequent neovascularization). Omental specimens were retrieved at different times posttransplantation (up to 180 days after transplant) and evaluation of the excised omentum with immunohistochemical staining for insulin and glucagon confirmed the presence of transplanted islets at each time point. These data provided evidence that islet autotransplantation within a VEGF-enriched fibrin gel resulted in engraftment at this site. Our initial preclinical experience with the omentum as an islet transplant site resulted in successful engraftment of syngeneic and allogeneic islets loaded on a synthetic biodegradable scaffold placed in an omental pouch in streptozocin-induced diabetic immunosuppressed cynomolgus monkeys.30 Animals that received allogeneic transplants underwent anti-IL-2 (daclizumab) induction with sirolimus and tacrolimus combination maintenance immunosuppression. All animals achieved C-peptide levels >1.0 ng/mL after transplantation, along with posttransplant decrease in exogenous insulin requirements and reduced HbA1c values. The levels of C-peptide achieved after transplantation were similar to those of the intrahepatic allogeneic islet recipients. However, there was a delay in graft function probably as
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a consequence of delayed vascularization and exposure of omental pouch islets to hypoxia during the early posttransplantation period. Omental graft explantation led to a rapid increase in exogenous insulin requirements, loss of C-peptide, and destabilization of metabolic control, suggesting that the transplanted islets were the source of C-peptide and responsible for the improvement in metabolic control. Histopathological analysis of the explanted grafts revealed the presence of well-granulated, well-vascularized insulin-positive islets surrounded by T cell subsets and macrophages, with minimal signs of lymphocyte infiltration.30 Building up on these results, we tested the feasibility of a novel approach of omental ITx. We engineered a biologic scaffold using plasma and recombinant human thrombin (rhT) in order to create a three-dimensional fibrin matrix trapping the islets and to promote islet graft adherence on the omental surface, prevent islet pelleting and support engraftment, neovascularization, and survival of the transplanted islets.31 This biologic scaffold was tested in a syngeneic and allogeneic diabetic rat model and in an allogeneic nonhuman primate model. The islet/plasma slurry was distributed onto the omental surface of treated animals and rhT was then gently dripped onto the graft, leading to an immediate gelling and adherence of the islets to the omental surface. Finally, the omentum was folded on itself in order to contain the graft and increase the contact area for the transplanted islets. Importantly, the biologic scaffold sustained allogeneic islet engraftment in immunosuppressed recipients, as supported by the achievement and maintenance of euglycemia during the follow-up period, along with the histopathological evidence of well-preserved islet cytoarchitecture, with abundant intragraft vascularization and positive insulin immunostaining after graft explantation.31
Clinical experience These findings provided preclinical evidence to proceed with a pilot phase I/II clinical trial. In 2014, we started enrolment for the clinical trial: “Allogeneic Islet Cells Transplanted Onto the Omentum” (ClinicalTrials. gov Identifier: NCT02213003) at our institution. This ongoing clinical trial is to test the feasibility and efficacy of ITx onto the omentum with a combined primary efficacy endpoint of HbA1c ≤6.5% at 1 year and absence of severe hypoglycemic events from day 28 to day 365 after ITx. The results for the first subject transplanted under this study have been previously reported.32 Briefly, it was a 43-year-old woman with a 25-year history of T1D complicated by hypoglycemia unawareness and episodes of severe hypoglycemia. She underwent ITx on the omentum with a total of 602,395 IEQ (11,386 IEQ/kg recipient body weight) obtained from a single deceased donor.
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Islets were combined with autologous plasma and gently layered on the omental surface through laparoscopic surgery. rhT was then layered over the islets-autologous plasma mix in order to generate a resorbable biologic scaffold. Lastly, the omentum was folded over the islet containing scaffold and additional rhT was used to seal the edges of the omental fold (Figs. 1–3). Induction immunosuppression was similar to the CIT-Phase 3 trial of ITx1 and consisted of antithymocyte globulin (ATG, Thymoglobulin) and etanercept (Enbrel), whereas maintenance immunosuppression consisted of tacrolimus and mycophenolate sodium. No surgical complications were reported. The patient achieved insulin independence 17 days after transplantation with excellent metabolic control. However, a functional decline was observed at 12 months, leading to insulin reintroduction by 15 months although with persistent graft function and stable glycemic control without hypoglycemia.33 We speculate that a change in immunosuppression (switching from tacrolimus to sirolimus due to the development of alopecia) may have led to the observed allograft dysfunction. The clinical outcomes on the two subsequent recipients have also been previously reported. Baseline characteristics and metabolic data for the three subjects are depicted in Table 1. Subject #2, a 32-year-old woman developed a hypersensitivity reaction following the third dose of ATG (prior to ITx), characterized by a diffuse erythematous rash comprising chest, abdomen extending to the groins, axillae, palms, and interdigital spaces, associated with facial flushing. There was no associated pruritus or hemodynamic instability. She was treated with high doses of IV corticosteroids resulting in improvement of the rash and then underwent ITx receiving 9635 IEQ/kg without complications. The rash resolved by day 3 post-ITx.
Thymoglobulin (ATG rabbit) is a purified, pasteurized, gamma immune globulin obtained by immunization of rabbits with human thymocytes and we hypothesize that the allergic reaction was likely related to possible preexisting recipient antirabbit antibodies. The induction course of ATG was completed as per study protocol although with pre-ATG infusion treatment with IV corticosteroids. Further, to prevent the development of serum sickness, the subject completed a 4-week course of high-dose prednisone post-ITx. Although the presence of C-peptide was demonstrated following ITx, stimulated C-peptide responses were marginal likely accounted by impaired engraftment and suboptimal effect of ATG related to the aforementioned hypersensitivity reaction. Despite this marginal C-peptide, subject maintained very good glycemic control evidenced by 14-day Dexcom G4 continuous glucose monitor (CGM) data obtained at 1 year posttransplant showing 88% time spent in the range 70–180 mg/dL, with an HbA1c of 5.6%. Nonetheless, subject did not meet the study primary efficacy outcome due to persistence of severe hypoglycemia and went on to receive an intrahepatic islet infusion as per protocol. Subject #3, a 46-year-old woman, received 12,648 IEQ/kg, resulting in marked metabolic improvement with a stimulated C-peptide of 2.47 ng/mL at 6 months. At 1 year post-ITx, insulin requirements had decreased by 51% compared to pre-ITx, HbA1c was 6.0% and there were no reported severe hypoglycemic episodes. Glycemic control was managed with the MiniMed 670G system and Guardian Sensor 3 CGM in Manual Mode. CGM data at 15 months post-ITx showed average sensor glucose of 113 ± 26 mg/dL with 96% time spent in glucose range 70–180 mg/dL, 3% time spent in glucose <70 mg/dL, and no time spent in glucose <55 mg/dL.
FIG. 1 Intraomental islet transplantation using a biologic resorbable scaffold. (1) The islets combined with the autologous plasma are layered on the omental surface. (2) Recombinant human thrombin is layered on top of the islet/plasma slurry, in order to create a three-dimensional fibrin matrix trapping the transplanted islets within the newly formed biologic scaffold. (3) The omentum is folded over in order to contain the graft and increase the contact area with the transplanted islets. (4) Additional thrombin is used to seal the omental fold.
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The greater omentum: A novel site for islet transplantation
FIG. 2 Islet pellet mixed with autologous plasma contained in a syringe. The red arrow shows the settled islet pellet at the bottom of the syringe. The blue arrow shows the autologous plasma above the islet pellet.
Most recently, Stice et al. reported on the results of combined site islet autotransplantation following total pancreatectomy using an omental pouch technique.34
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Notably, patients who developed intraoperative complications (i.e., increased portal pressures and infusion catheter displacement) precluding the complete intrahepatic islet infusion underwent implantation of the remaining islet mass within an omental pouch. A fibrin sealant consisting of human fibrinogen, fibronectin, factor XIII, and human thrombin (Evicel, Johnson & Johnson Wound Management, Somerville, NJ) was applied over the islets to sustain their adhesion to the omentum and to promote the engraftment. The omental pouch was then closed using a running suture. Although all the intraomental islet recipients had partial graft function posttransplant (assessed by C-peptide levels), no significant differences in glycemic control or graft function were observed at 3-month follow-up with respect to patients receiving intrahepatic islet transplant alone. The authors noted that the short follow-up period and sample size may limit interpretation of their data. Our initial experience has demonstrated feasibility and safety of allo-ITx on the omentum with persistent graft function throughout follow-up, resulting in improved metabolic control and absence of severe hypoglycemia (subjects #1 and #3). However, metabolic results were lower than expected for the transplanted islet mass, suggesting a marked loss of islets early postITx. Inadequate nutrient and oxygen delivery during the days prior to graft revascularization likely play a major role in islet engraftment at the omentum site.
FIG. 3 Laparoscopic view of islet transplantation on the omentum. Panel 1 shows the omentum being identified and spread flat over the intestinal loops prior to delivery of the islet product. Panel 2 shows the islet mix layered as droplets onto the omentum surface. Panel 3 shows the addition of thrombin to the islets. Panel 4 shows the islets in the biologic scaffold as light tan patches spread over the omentum surface (red arrows). Panel 5 shows the folding of the omentum over the islets. Panel 6 shows the omentum folded at the end of the procedure.
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MMTTb Stimulated glucose (mg/dL)/ stimulated C-peptide (ng/mL)
Insulin dose IU/kg/day (IU/day)
Subject #
Gender
Age (years)
T1D Duration (years)
1
Female
43
26
21.5
11,386
181/3.32
277/1.79
0.62 (33)
0
6.8
5.4
2
Female
32
16
25.3
9635
372/0.88
374/0.65
0.45 (31)
0.44 (29)
5.7
5.6
3
Female
46
42
24
12,648
277/2.47
261/1.01
0.45 (20)
0.23 (13)
6.3
6.0
a
IEQ = islet equivalents. MMTT = mixed meal tolerance test. c ITx = islet transplantation. b
HbA1c (%)
BMI (kg/m2)
IEQ /kg
6 months
12 months
Pre-ITx
12 months
Pre-ITx
12 months
a
c
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TABLE 1 Baseline characteristics and metabolic data up to 1 year posttransplantation
References
Further, the timing for full islet allograft revascularization at this site is currently unknown. In addition, the local inflammatory profile elicited by transplantation at this site remains uncharacterized. Novel strategies to improve oxygen delivery and promote graft neovascularization are needed to potentially improve engraftment and long-term outcomes. Our results, however, need to be interpreted cautiously as they are confounded by the development of adverse events on the transplanted subjects requiring interventions that may have impacted engraftment and metabolic outcomes. Thus, additional transplants are needed to adequately assess this transplant site. Other investigators are also currently exploring the omentum site for ITx in patients with T1D (ClinicalTrials.gov Identifiers: NCT02803905).
Conclusions In summary, apart from the intrahepatic site, the omentum is the only site that has shown long-term islet engraftment and function. In view of this and considering the unique advantages offered by an extravascular transplantation site, further evaluation is warranted. Additional transplants are planned under our current clinical trial and longer follow-up will allow to more extensively evaluate this site. In parallel, the development of strategies to improve local oxygen delivery, promote graft neovascularization, and minimize immunosuppression will be required to improve long-term outcomes at this novel transplant site.
References 1. Hering BJ, Clarke WR, Bridges ND, et al. Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care. 2016;39(7):1230–1240. 2. Rickels MR, Stock PG, de Koning EJP, et al. Defining outcomes for beta-cell replacement therapy in the treatment of diabetes: a consensus report on the igls criteria from the IPITA/EPITA opinion leaders workshop. Transplantation. 2018;102(9):1479–1486. 3. Lablanche S, Vantyghem MC, Kessler L, et al. Islet transplantation versus insulin therapy in patients with type 1 diabetes with severe hypoglycaemia or poorly controlled glycaemia after kidney transplantation (TRIMECO): a multicentre, randomised controlled trial. Lancet Diabetes Endocrinol. 2018;6(7):527–537. 4. Brendel MD, Hering BJ, Schultz AO, Bretzel RG. International Islet Transplant Registry, Newsletter #9. https://www.med.uni-giessen.de/ itr/newsletter/no_9/news_9.pdf; 2001. (Accessed March 29, 2019). 5. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343(4):230–238. 6. Froud T, Ricordi C, Baidal DA, et al. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience. Am J Transplant. 2005;5(8):2037–2046. 7. Bellin MD, Barton FB, Heitman A, et al. Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type 1 diabetes. Am J Transplant. 2012;12(6):1576–1583.
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8. Collaborative Islet Transplant Registry. Collaborative Islet Transplant Registry Tenth Annual Report. https://citregistry.org/system/ files/10th_AR.pdf; 2017. (Accessed September 15, 2018). 9. Merani S, Toso C, Emamaullee J, Shapiro AM. Optimal implantation site for pancreatic islet transplantation. Br J Surg. 2008;95(12):1449–1461. 10. Cantarelli E, Piemonti L. Alternative transplantation sites for pancreatic islet grafts. Curr Diab Rep. 2011;11(5):364–374. 11. Casey JJ, Lakey JR, Ryan EA, et al. Portal venous pressure changes after sequential clinical islet transplantation. Transplantation. 2002;74(7):913–915. 12. Venturini M, Angeli E, Maffi P, et al. Technique, complications, and therapeutic efficacy of percutaneous transplantation of human pancreatic islet cells in type 1 diabetes: the role of US. Radiology. 2005;234(2):617–624. 13. Bennet W, Groth CG, Larsson R, Nilsson B, Korsgren O. Isolated human islets trigger an instant blood mediated inflammatory reaction: implications for intraportal islet transplantation as a treatment for patients with type 1 diabetes. Ups J Med Sci. 2000;105(2):125–133. 14. Yin D, Ding JW, Shen J, Ma L, Hara M, Chong AS. Liver ischemia contributes to early islet failure following intraportal transplantation: benefits of liver ischemic-preconditioning. Am J Transplant. 2006;6(1):60–68. 15. Bottino R, Fernandez LA, Ricordi C, et al. Transplantation of allogeneic islets of Langerhans in the rat liver: effects of macrophage depletion on graft survival and microenvironment activation. Diabetes. 1998;47(3):316–323. 16. Barshes NR, Wyllie S, Goss JA. Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts. J Leukoc Biol. 2005;77(5):587–597. 17. Sakata N, Hayes P, Tan A, et al. MRI assessment of ischemic liver after intraportal islet transplantation. Transplantation. 2009;87(6):825–830. 18. Cantaluppi V, Biancone L, Romanazzi GM, et al. Antiangiogenic and immunomodulatory effects of rapamycin on islet endothelium: relevance for islet transplantation. Am J Transplant. 2006;6(11):2601–2611. 19. Shapiro AM, Gallant HL, Hao EG, et al. The portal immunosuppressive storm: relevance to islet transplantation? Ther Drug Monit. 2005;27(1):35–37. 20. Jones GL, Juszczak MT, Hughes SJ, Kooner P, Powis SH, Press M. Time course and quantification of pancreatic islet revascularization following intraportal transplantation. Cell Transplant. 2007;16(5):505–516. 21. Davalli AM, Scaglia L, Zangen DH, Hollister J, Bonner-Weir S, Weir GC. Vulnerability of islets in the immediate posttransplantation period. Dynamic changes in structure and function. Diabetes. 1996;45(9):1161–1167. 22. Tze WJ, Tai J. Immunological studies in diabetic rat recipients with a pancreatic islet cell allograft in the brain. Transplantation. 1989;47(6):1053–1057. 23. Pellicciaro VI M, Lanzoni G, Tisone G, Ricordi G. The greater omentum as a site for pancreatic islet transplantation. CellR4. 2017;5(3):e2418. 24. Litbarg NO, Gudehithlu KP, Sethupathi P, Arruda JA, Dunea G, Singh AK. Activated omentum becomes rich in factors that promote healing and tissue regeneration. Cell Tissue Res. 2007;328(3):487–497. 25. Ao Z, Matayoshi K, Lakey JR, Rajotte RV, Warnock GL. Survival and function of purified islets in the omental pouch site of outbred dogs. Transplantation. 1993;56(3):524–529. 26. Gustavson SM, Rajotte RV, Hunkeler D, et al. Islet auto- transplantation into an omental or splenic site results in a normal beta cell but abnormal alpha cell response to mild non-insulin- induced hypoglycemia. Am J Transplant. 2005;5(10):2368–2377.
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27. Kin T, Korbutt GS, Rajotte RV. Survival and metabolic function of syngeneic rat islet grafts transplanted in the omental pouch. Am J Transplant. 2003;3(3):281–285. 28. Kobayashi T, Aomatsu Y, Iwata H, et al. Indefinite islet protection from autoimmune destruction in nonobese diabetic mice by agarose microencapsulation without immunosuppression. Transplantation. 2003;75(5):619–625. 29. Hefty TR, Kuhr CS, Chong KT, et al. Omental roll-up: a technique for islet engraftment in a large animal model. J Surg Res. 2010;161(1):134–138. 30. Berman DM, O'Neil JJ, Coffey LC, et al. Long-term survival of nonhuman primate islets implanted in an omental pouch on a biodegradable scaffold. Am J Transplant. 2009;9(1):91–104.
31. Berman DM, Molano RD, Fotino C, et al. Bioengineering the endocrine pancreas: intraomental islet transplantation within a biologic resorbable scaffold. Diabetes. 2016;65(5):1350–1361. 32. Baidal DA, Ricordi C, Berman DM, et al. Bioengineering of an intraabdominal endocrine pancreas. N Engl J Med. 2017;376(19):1887–1889. 33. D. Baidal, C. Ricordi, D.M. Berman, et al., Long-term function of islet allografts transplanted on the omentum using a biological scaffold. Oral presentation at American Diabetes Association 78th Scientific Sessions, June 23, 2018. 34. Stice MJ, Dunn TB, Bellin MD, Skube ME, Beilman GJ. Omental pouch technique for combined site islet autotransplantation following total pancreatectomy. Cell Transplant. 2018;27(10):1561–1568.
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19 A biologic resorbable scaffold for tissue engineering of the endocrine pancreas: Clinical experience of islet transplantation on the omentum David A. Baidal⁎,†, Marco Infante⁎,‡, Virginia Fuenmayor⁎, Ana M. Alvarez⁎, Nathalia Padilla⁎, Dora M. Berman⁎,‡, Antonello Pileggi¶, Elina Linetsky⁎,§, Gaetano Ciancio§, Camillo Ricordi⁎,§,ǁ, Rodolfo Alejandro⁎,† ⁎
Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States †Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, United States ‡Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy §Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States ¶Division of Physiology and Pathological Sciences, Center for Scientific Review, National Institutes of Health, Bethesda, MD, United States ǁcGMP Facility, Cell Transplant Center, Division of Cellular Transplant, Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States
O U T L I N E Introduction: The intrahepatic site for islet transplantation
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Extrahepatic sites for islet transplantation
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The greater omentum: A novel site for islet transplantation
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Introduction: The intrahepatic site for islet transplantation Islet transplantation (ITx) is a cellular therapy that has demonstrated efficacy in improving glycemic control and abolishing hypoglycemia in patients affected by type 1 diabetes (T1D) complicated by hypoglycemia unawareness and episodes of severe hypoglycemia.1–3 However, it has been a long road prior to achieving the success rates that are currently observed with ITx. Prior Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00019-1
Preclinical experience Clinical experience
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Conclusions
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References
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to 2000, ITx efforts throughout centers worldwide were met with very limited function evidenced by <10% of patients achieving insulin independence by 1 year posttransplantation.4 It was not until the introduction of the Edmonton protocol in 2000 that intrahepatic ITx really surfaced as a successful therapeutic modality for adult patients affected by T1D.5 Shapiro et al. reported insulin independence at 1 year on seven consecutive subjects, a breakthrough for the field at the time.5 The key factors associated with the success of this novel transplantation
269
© 2020 Elsevier Inc. All rights reserved.
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protocol included the intraportal infusion of a cumulative islet mass of ≥10,000 islet equivalents (IEQ) per kg of body weight obtained from at least two islet infusions, the avoidance of glucocorticoids, induction immunosuppression with IL-2 blockade, and maintenance immunosuppression with the combination of an mTOR inhibitor (sirolimus) and an FK-506 inhibitor (tacrolimus). However, a particular limitation of this protocol was the need to transplant the islets within 4 h from isolation which precluded a more thorough assessment of the islet preparation prior to release for transplantation. The Miami group showed similar success rates using an “islets in culture” protocol compared with the initial Edmonton experience.6 Culturing of islets allowed for additional assessment of the islet product prior to transplantation and for better determination whether the islet product was suitable for clinical transplantation. In 2012, the use of potent induction immunotherapy with thymoglobulin combined with TNF-α blockade demonstrated improved outcomes at 5 years, similar to results observed in pancreas transplantation.7 Despite these improvements in immunosuppressive and antiinflammatory strategies, long-term data of intrahepatic islet allografts show a progressive decline in beta-cell function.8 Several factors may contribute to this loss, including allograft rejection, recurrence of autoimmunity, and metabolic exhaustion.9, 10 In addition, the use of an intravascular site poses several challenges. Intraportal islet infusion is associated with an increase in portal vein pressure that is proportional to the volume and number of the transplanted islets, thus limiting the total islet mass that can be transplanted.11, 12 Other limitations of the intraportal infusion include the potential risk of bleeding and thrombosis.12 In addition, an instant blood-mediated inflammatory reaction (IBMIR) is triggered once islets come into contact with blood.13 The IBMIR results in the activation of the coagulation and complement cascade, leading to formation of clots around the islets which contribute to a significant loss of islet integrity and function.14 Moreover, intrahepatic islet infusion leads to thrombosis and liver ischemia due to islet entrapment in the liver sinusoids, thus resulting in the activation of sinusoidal endothelial cells and Kupffer cells with subsequent islet cell damage and dysfunction.15, 16 In animal models, all the aforementioned ischemia-related events have been shown to result in the loss of approximately 60% of intrahepatic islets due to necrosis and apoptosis observed during the first days posttransplantation.14, 17 The direct exposure of the islet graft to immunosuppressive drugs, nutrients, gut hormones, and metabolites directly from the portal circulation is another important aspect to consider as this may contribute to graft dysfunction.10, 18 As orally ingested immunosuppressive drugs enter the portal circulation, they come into contact with the transplanted islets before undergoing first-pass metabolism.
This leads to the intrahepatic islets being exposed to immunosuppressive drug levels that are higher compared to the systemic circulation, which in turn may result in enhancing drug-related toxic effects on islet function.18, 19 Lastly, revascularization of intraportally transplanted islets takes approximately 7–14 days during which time oxygenation—which is provided by both the portal system and diffusion from the hepatic artery circulation— remains suboptimal, compromising engraftment and aggravating beta cell loss.20, 21 In conclusion, although the intrahepatic site is the only clinical site that has shown islet allograft engraftment with achievement of insulin-independence and long-term graft function, it remains far from being an ideal transplantation site.
Extrahepatic sites for islet transplantation In light of the aforementioned limitations, the identification of an “extrahepatic” site has thus been pursued in the islet transplant field to improve graft engraftment and survival. The optimal characteristics for an ITx site are as follows: • ability to accommodate an adequate islet mass • accessibility by minimally invasive transplant procedures • avoidance or minimization of immune and inflammatory responses • adequate arterial vascular network for nutrient and oxygen delivery • physiologic venous drainage through the portal system • allowing for noninvasive longitudinal morphological and functional monitoring of the islets posttransplants • accessibility for graft biopsy Several preclinical studies have investigated a series of extrahepatic sites for ITx, including pancreas, gastric submucosa, genitourinary tract (i.e., subcapsular renal site, bladder submucosa, and testis), skeletal muscle, subcutaneous space, peritoneal cavity, omentum, spleen, bone marrow, anterior chamber of the eye, thymus, bone marrow, and central nervous system.10, 22 A few of them have been translated to the clinical arena, but have been met with minimal success with the exception of the omentum.
The greater omentum: A novel site for islet transplantation The greater omentum is a highly vascularized structure located in the peritoneal cavity and composed of connective, adipose, and vascular cells, along with specialized immune cells. It has long been considered as an
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The greater omentum: A novel site for islet transplantation
appealing site for ITx as it offers a large and highly vascularized surface area (which may facilitate diffusion of nutrients and oxygen to the graft early after transplantation), exclusive portal drainage, and easy accessibility by minimally invasive procedures.23 Moreover, it displays a remarkable angiogenic potential in response to mechanical and ischemic injury, which may favor islet engraftment and survival.24
Preclinical experience The safety and efficacy of islet allo- and autotransplantation in an omental pouch have been previously demonstrated in preclinical models. Studies in dogs demonstrated that islets infused into an omental pouch restored normoglycemia in pancreatectomized recipients.25, 26 Similarly, Kin et al. found that syngeneic islets transplanted into an omental pouch restored normoglycemia in streptozotocin-induced diabetic rats and nonobese diabetic (NOD) mice.27, 28 Hefty et al. investigated the efficacy of a novel surgical technique for intraomental ITx in dogs.29 The islet pellet was resuspended in phosphate-buffered saline (PBS) containing vascular endothelial growth factor (VEGF). Autologous plasma was then mixed with the islet/VEGF suspension and the resulting coagulum placed onto the omental ventral surface of recipient dogs that previously underwent partial or total pancreatectomy. Then, the leading edge of the greater omentum was rolled up in order to secure the transplanted islets in position and maximize the contact area of the thin islet layer with the two omental surfaces (for nutrient diffusion and subsequent neovascularization). Omental specimens were retrieved at different times posttransplantation (up to 180 days after transplant) and evaluation of the excised omentum with immunohistochemical staining for insulin and glucagon confirmed the presence of transplanted islets at each time point. These data provided evidence that islet autotransplantation within a VEGF-enriched fibrin gel resulted in engraftment at this site. Our initial preclinical experience with the omentum as an islet transplant site resulted in successful engraftment of syngeneic and allogeneic islets loaded on a synthetic biodegradable scaffold placed in an omental pouch in streptozocin-induced diabetic immunosuppressed cynomolgus monkeys.30 Animals that received allogeneic transplants underwent anti-IL-2 (daclizumab) induction with sirolimus and tacrolimus combination maintenance immunosuppression. All animals achieved C-peptide levels >1.0 ng/mL after transplantation, along with posttransplant decrease in exogenous insulin requirements and reduced HbA1c values. The levels of C-peptide achieved after transplantation were similar to those of the intrahepatic allogeneic islet recipients. However, there was a delay in graft function probably as
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a consequence of delayed vascularization and exposure of omental pouch islets to hypoxia during the early posttransplantation period. Omental graft explantation led to a rapid increase in exogenous insulin requirements, loss of C-peptide, and destabilization of metabolic control, suggesting that the transplanted islets were the source of C-peptide and responsible for the improvement in metabolic control. Histopathological analysis of the explanted grafts revealed the presence of well-granulated, well-vascularized insulin-positive islets surrounded by T cell subsets and macrophages, with minimal signs of lymphocyte infiltration.30 Building up on these results, we tested the feasibility of a novel approach of omental ITx. We engineered a biologic scaffold using plasma and recombinant human thrombin (rhT) in order to create a three-dimensional fibrin matrix trapping the islets and to promote islet graft adherence on the omental surface, prevent islet pelleting and support engraftment, neovascularization, and survival of the transplanted islets.31 This biologic scaffold was tested in a syngeneic and allogeneic diabetic rat model and in an allogeneic nonhuman primate model. The islet/plasma slurry was distributed onto the omental surface of treated animals and rhT was then gently dripped onto the graft, leading to an immediate gelling and adherence of the islets to the omental surface. Finally, the omentum was folded on itself in order to contain the graft and increase the contact area for the transplanted islets. Importantly, the biologic scaffold sustained allogeneic islet engraftment in immunosuppressed recipients, as supported by the achievement and maintenance of euglycemia during the follow-up period, along with the histopathological evidence of well-preserved islet cytoarchitecture, with abundant intragraft vascularization and positive insulin immunostaining after graft explantation.31
Clinical experience These findings provided preclinical evidence to proceed with a pilot phase I/II clinical trial. In 2014, we started enrolment for the clinical trial: “Allogeneic Islet Cells Transplanted Onto the Omentum” (ClinicalTrials. gov Identifier: NCT02213003) at our institution. This ongoing clinical trial is to test the feasibility and efficacy of ITx onto the omentum with a combined primary efficacy endpoint of HbA1c ≤6.5% at 1 year and absence of severe hypoglycemic events from day 28 to day 365 after ITx. The results for the first subject transplanted under this study have been previously reported.32 Briefly, it was a 43-year-old woman with a 25-year history of T1D complicated by hypoglycemia unawareness and episodes of severe hypoglycemia. She underwent ITx on the omentum with a total of 602,395 IEQ (11,386 IEQ/kg recipient body weight) obtained from a single deceased donor.
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Islets were combined with autologous plasma and gently layered on the omental surface through laparoscopic surgery. rhT was then layered over the islets-autologous plasma mix in order to generate a resorbable biologic scaffold. Lastly, the omentum was folded over the islet containing scaffold and additional rhT was used to seal the edges of the omental fold (Figs. 1–3). Induction immunosuppression was similar to the CIT-Phase 3 trial of ITx1 and consisted of antithymocyte globulin (ATG, Thymoglobulin) and etanercept (Enbrel), whereas maintenance immunosuppression consisted of tacrolimus and mycophenolate sodium. No surgical complications were reported. The patient achieved insulin independence 17 days after transplantation with excellent metabolic control. However, a functional decline was observed at 12 months, leading to insulin reintroduction by 15 months although with persistent graft function and stable glycemic control without hypoglycemia.33 We speculate that a change in immunosuppression (switching from tacrolimus to sirolimus due to the development of alopecia) may have led to the observed allograft dysfunction. The clinical outcomes on the two subsequent recipients have also been previously reported. Baseline characteristics and metabolic data for the three subjects are depicted in Table 1. Subject #2, a 32-year-old woman developed a hypersensitivity reaction following the third dose of ATG (prior to ITx), characterized by a diffuse erythematous rash comprising chest, abdomen extending to the groins, axillae, palms, and interdigital spaces, associated with facial flushing. There was no associated pruritus or hemodynamic instability. She was treated with high doses of IV corticosteroids resulting in improvement of the rash and then underwent ITx receiving 9635 IEQ/kg without complications. The rash resolved by day 3 post-ITx.
Thymoglobulin (ATG rabbit) is a purified, pasteurized, gamma immune globulin obtained by immunization of rabbits with human thymocytes and we hypothesize that the allergic reaction was likely related to possible preexisting recipient antirabbit antibodies. The induction course of ATG was completed as per study protocol although with pre-ATG infusion treatment with IV corticosteroids. Further, to prevent the development of serum sickness, the subject completed a 4-week course of high-dose prednisone post-ITx. Although the presence of C-peptide was demonstrated following ITx, stimulated C-peptide responses were marginal likely accounted by impaired engraftment and suboptimal effect of ATG related to the aforementioned hypersensitivity reaction. Despite this marginal C-peptide, subject maintained very good glycemic control evidenced by 14-day Dexcom G4 continuous glucose monitor (CGM) data obtained at 1 year posttransplant showing 88% time spent in the range 70–180 mg/dL, with an HbA1c of 5.6%. Nonetheless, subject did not meet the study primary efficacy outcome due to persistence of severe hypoglycemia and went on to receive an intrahepatic islet infusion as per protocol. Subject #3, a 46-year-old woman, received 12,648 IEQ/kg, resulting in marked metabolic improvement with a stimulated C-peptide of 2.47 ng/mL at 6 months. At 1 year post-ITx, insulin requirements had decreased by 51% compared to pre-ITx, HbA1c was 6.0% and there were no reported severe hypoglycemic episodes. Glycemic control was managed with the MiniMed 670G system and Guardian Sensor 3 CGM in Manual Mode. CGM data at 15 months post-ITx showed average sensor glucose of 113 ± 26 mg/dL with 96% time spent in glucose range 70–180 mg/dL, 3% time spent in glucose <70 mg/dL, and no time spent in glucose <55 mg/dL.
FIG. 1 Intraomental islet transplantation using a biologic resorbable scaffold. (1) The islets combined with the autologous plasma are layered on the omental surface. (2) Recombinant human thrombin is layered on top of the islet/plasma slurry, in order to create a three-dimensional fibrin matrix trapping the transplanted islets within the newly formed biologic scaffold. (3) The omentum is folded over in order to contain the graft and increase the contact area with the transplanted islets. (4) Additional thrombin is used to seal the omental fold.
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The greater omentum: A novel site for islet transplantation
FIG. 2 Islet pellet mixed with autologous plasma contained in a syringe. The red arrow shows the settled islet pellet at the bottom of the syringe. The blue arrow shows the autologous plasma above the islet pellet.
Most recently, Stice et al. reported on the results of combined site islet autotransplantation following total pancreatectomy using an omental pouch technique.34
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Notably, patients who developed intraoperative complications (i.e., increased portal pressures and infusion catheter displacement) precluding the complete intrahepatic islet infusion underwent implantation of the remaining islet mass within an omental pouch. A fibrin sealant consisting of human fibrinogen, fibronectin, factor XIII, and human thrombin (Evicel, Johnson & Johnson Wound Management, Somerville, NJ) was applied over the islets to sustain their adhesion to the omentum and to promote the engraftment. The omental pouch was then closed using a running suture. Although all the intraomental islet recipients had partial graft function posttransplant (assessed by C-peptide levels), no significant differences in glycemic control or graft function were observed at 3-month follow-up with respect to patients receiving intrahepatic islet transplant alone. The authors noted that the short follow-up period and sample size may limit interpretation of their data. Our initial experience has demonstrated feasibility and safety of allo-ITx on the omentum with persistent graft function throughout follow-up, resulting in improved metabolic control and absence of severe hypoglycemia (subjects #1 and #3). However, metabolic results were lower than expected for the transplanted islet mass, suggesting a marked loss of islets early postITx. Inadequate nutrient and oxygen delivery during the days prior to graft revascularization likely play a major role in islet engraftment at the omentum site.
FIG. 3 Laparoscopic view of islet transplantation on the omentum. Panel 1 shows the omentum being identified and spread flat over the intestinal loops prior to delivery of the islet product. Panel 2 shows the islet mix layered as droplets onto the omentum surface. Panel 3 shows the addition of thrombin to the islets. Panel 4 shows the islets in the biologic scaffold as light tan patches spread over the omentum surface (red arrows). Panel 5 shows the folding of the omentum over the islets. Panel 6 shows the omentum folded at the end of the procedure.
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MMTTb Stimulated glucose (mg/dL)/ stimulated C-peptide (ng/mL)
Insulin dose IU/kg/day (IU/day)
Subject #
Gender
Age (years)
T1D Duration (years)
1
Female
43
26
21.5
11,386
181/3.32
277/1.79
0.62 (33)
0
6.8
5.4
2
Female
32
16
25.3
9635
372/0.88
374/0.65
0.45 (31)
0.44 (29)
5.7
5.6
3
Female
46
42
24
12,648
277/2.47
261/1.01
0.45 (20)
0.23 (13)
6.3
6.0
a
IEQ = islet equivalents. MMTT = mixed meal tolerance test. c ITx = islet transplantation. b
HbA1c (%)
BMI (kg/m2)
IEQ /kg
6 months
12 months
Pre-ITx
12 months
Pre-ITx
12 months
a
c
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TABLE 1 Baseline characteristics and metabolic data up to 1 year posttransplantation
References
Further, the timing for full islet allograft revascularization at this site is currently unknown. In addition, the local inflammatory profile elicited by transplantation at this site remains uncharacterized. Novel strategies to improve oxygen delivery and promote graft neovascularization are needed to potentially improve engraftment and long-term outcomes. Our results, however, need to be interpreted cautiously as they are confounded by the development of adverse events on the transplanted subjects requiring interventions that may have impacted engraftment and metabolic outcomes. Thus, additional transplants are needed to adequately assess this transplant site. Other investigators are also currently exploring the omentum site for ITx in patients with T1D (ClinicalTrials.gov Identifiers: NCT02803905).
Conclusions In summary, apart from the intrahepatic site, the omentum is the only site that has shown long-term islet engraftment and function. In view of this and considering the unique advantages offered by an extravascular transplantation site, further evaluation is warranted. Additional transplants are planned under our current clinical trial and longer follow-up will allow to more extensively evaluate this site. In parallel, the development of strategies to improve local oxygen delivery, promote graft neovascularization, and minimize immunosuppression will be required to improve long-term outcomes at this novel transplant site.
References 1. Hering BJ, Clarke WR, Bridges ND, et al. Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care. 2016;39(7):1230–1240. 2. Rickels MR, Stock PG, de Koning EJP, et al. Defining outcomes for beta-cell replacement therapy in the treatment of diabetes: a consensus report on the igls criteria from the IPITA/EPITA opinion leaders workshop. Transplantation. 2018;102(9):1479–1486. 3. Lablanche S, Vantyghem MC, Kessler L, et al. Islet transplantation versus insulin therapy in patients with type 1 diabetes with severe hypoglycaemia or poorly controlled glycaemia after kidney transplantation (TRIMECO): a multicentre, randomised controlled trial. Lancet Diabetes Endocrinol. 2018;6(7):527–537. 4. Brendel MD, Hering BJ, Schultz AO, Bretzel RG. International Islet Transplant Registry, Newsletter #9. https://www.med.uni-giessen.de/ itr/newsletter/no_9/news_9.pdf; 2001. (Accessed March 29, 2019). 5. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343(4):230–238. 6. Froud T, Ricordi C, Baidal DA, et al. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience. Am J Transplant. 2005;5(8):2037–2046. 7. Bellin MD, Barton FB, Heitman A, et al. Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type 1 diabetes. Am J Transplant. 2012;12(6):1576–1583.
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8. Collaborative Islet Transplant Registry. Collaborative Islet Transplant Registry Tenth Annual Report. https://citregistry.org/system/ files/10th_AR.pdf; 2017. (Accessed September 15, 2018). 9. Merani S, Toso C, Emamaullee J, Shapiro AM. Optimal implantation site for pancreatic islet transplantation. Br J Surg. 2008;95(12):1449–1461. 10. Cantarelli E, Piemonti L. Alternative transplantation sites for pancreatic islet grafts. Curr Diab Rep. 2011;11(5):364–374. 11. Casey JJ, Lakey JR, Ryan EA, et al. Portal venous pressure changes after sequential clinical islet transplantation. Transplantation. 2002;74(7):913–915. 12. Venturini M, Angeli E, Maffi P, et al. Technique, complications, and therapeutic efficacy of percutaneous transplantation of human pancreatic islet cells in type 1 diabetes: the role of US. Radiology. 2005;234(2):617–624. 13. Bennet W, Groth CG, Larsson R, Nilsson B, Korsgren O. Isolated human islets trigger an instant blood mediated inflammatory reaction: implications for intraportal islet transplantation as a treatment for patients with type 1 diabetes. Ups J Med Sci. 2000;105(2):125–133. 14. Yin D, Ding JW, Shen J, Ma L, Hara M, Chong AS. Liver ischemia contributes to early islet failure following intraportal transplantation: benefits of liver ischemic-preconditioning. Am J Transplant. 2006;6(1):60–68. 15. Bottino R, Fernandez LA, Ricordi C, et al. Transplantation of allogeneic islets of Langerhans in the rat liver: effects of macrophage depletion on graft survival and microenvironment activation. Diabetes. 1998;47(3):316–323. 16. Barshes NR, Wyllie S, Goss JA. Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts. J Leukoc Biol. 2005;77(5):587–597. 17. Sakata N, Hayes P, Tan A, et al. MRI assessment of ischemic liver after intraportal islet transplantation. Transplantation. 2009;87(6):825–830. 18. Cantaluppi V, Biancone L, Romanazzi GM, et al. Antiangiogenic and immunomodulatory effects of rapamycin on islet endothelium: relevance for islet transplantation. Am J Transplant. 2006;6(11):2601–2611. 19. Shapiro AM, Gallant HL, Hao EG, et al. The portal immunosuppressive storm: relevance to islet transplantation? Ther Drug Monit. 2005;27(1):35–37. 20. Jones GL, Juszczak MT, Hughes SJ, Kooner P, Powis SH, Press M. Time course and quantification of pancreatic islet revascularization following intraportal transplantation. Cell Transplant. 2007;16(5):505–516. 21. Davalli AM, Scaglia L, Zangen DH, Hollister J, Bonner-Weir S, Weir GC. Vulnerability of islets in the immediate posttransplantation period. Dynamic changes in structure and function. Diabetes. 1996;45(9):1161–1167. 22. Tze WJ, Tai J. Immunological studies in diabetic rat recipients with a pancreatic islet cell allograft in the brain. Transplantation. 1989;47(6):1053–1057. 23. Pellicciaro VI M, Lanzoni G, Tisone G, Ricordi G. The greater omentum as a site for pancreatic islet transplantation. CellR4. 2017;5(3):e2418. 24. Litbarg NO, Gudehithlu KP, Sethupathi P, Arruda JA, Dunea G, Singh AK. Activated omentum becomes rich in factors that promote healing and tissue regeneration. Cell Tissue Res. 2007;328(3):487–497. 25. Ao Z, Matayoshi K, Lakey JR, Rajotte RV, Warnock GL. Survival and function of purified islets in the omental pouch site of outbred dogs. Transplantation. 1993;56(3):524–529. 26. Gustavson SM, Rajotte RV, Hunkeler D, et al. Islet auto- transplantation into an omental or splenic site results in a normal beta cell but abnormal alpha cell response to mild non-insulin- induced hypoglycemia. Am J Transplant. 2005;5(10):2368–2377.
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27. Kin T, Korbutt GS, Rajotte RV. Survival and metabolic function of syngeneic rat islet grafts transplanted in the omental pouch. Am J Transplant. 2003;3(3):281–285. 28. Kobayashi T, Aomatsu Y, Iwata H, et al. Indefinite islet protection from autoimmune destruction in nonobese diabetic mice by agarose microencapsulation without immunosuppression. Transplantation. 2003;75(5):619–625. 29. Hefty TR, Kuhr CS, Chong KT, et al. Omental roll-up: a technique for islet engraftment in a large animal model. J Surg Res. 2010;161(1):134–138. 30. Berman DM, O'Neil JJ, Coffey LC, et al. Long-term survival of nonhuman primate islets implanted in an omental pouch on a biodegradable scaffold. Am J Transplant. 2009;9(1):91–104.
31. Berman DM, Molano RD, Fotino C, et al. Bioengineering the endocrine pancreas: intraomental islet transplantation within a biologic resorbable scaffold. Diabetes. 2016;65(5):1350–1361. 32. Baidal DA, Ricordi C, Berman DM, et al. Bioengineering of an intraabdominal endocrine pancreas. N Engl J Med. 2017;376(19):1887–1889. 33. D. Baidal, C. Ricordi, D.M. Berman, et al., Long-term function of islet allografts transplanted on the omentum using a biological scaffold. Oral presentation at American Diabetes Association 78th Scientific Sessions, June 23, 2018. 34. Stice MJ, Dunn TB, Bellin MD, Skube ME, Beilman GJ. Omental pouch technique for combined site islet autotransplantation following total pancreatectomy. Cell Transplant. 2018;27(10):1561–1568.
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