Development of an Immunoisolated Endovascular Islet Cell Stent to Treat Type I Diabetes Mellitus  

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Gaston, Varun Singh, Anil Kharga, Kevin Deng, Kang Mi Lee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6441612/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction and Objectives: Despite recent advances in cell sources and immunosuppression protocols, islet cell transplantation has had limited clinical success in achieving long-term freedom from exogenous insulin therapy for individuals with type 1 diabetes. Currently, islet cell transplants are performed via the infusion of islet cells into the recipient's portal vein. Approximately 60% of transplanted cells die within 3 days of transplantation due to ischemic injury. 7 Significant immunosuppression is needed to avoid rejection but is also toxic to islet cells, and the use of immunosuppression remains a barrier to more widespread use of islet cell transplantation. To address these challenges, we developed an endovascular biologic stent-graft, IsletStent, which is designed to utilize semipermeable membranes to protect transplanted islets from immune cell attack and prevent ischemic injury through endovascular deployment. Methods: Semipermeable cell chambers in stent-grafts were fabricated from ePTFE with a pore size of 0.22 µm. The cell chamber was seeded with islet cells harvested from 6–8-week-old male BALB/c mice (Jackson Laboratories, Bar Harbor, ME). Human islets were obtained courtesy of an institutional islet isolation GMP facility. A benchtop normothermic machine perfusion circuit was used to model intravascular deployment. Within the perfusion circuit, devices were exposed to varying glucose concentrations in the perfusion media, and samples were drawn for insulin analysis. Insulin levels were measured via ELISA. Results: In murine islet cells, there were significantly increased levels of insulin secretion after exposure to high-glucose media. After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 83 ± 8 mIU/ml, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 145 ± 20 mIU/ml. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.02; α = 0.05). The rate of insulin secretion was significantly greater under high-glucose conditions than under low-glucose conditions (6759 ± 503 mIU/h vs. 5344 ± 144 mIU/hr, p = 0.008, α = 0.05). After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 229 ± 13 mIU/dL, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 300 ± 17 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.014; α = 0.05). Conclusions: The IsletStent is a novel biologic endovascular device that enables islet cell function and survival within a semipermeable cell chamber placed intravascularly. However, further in-vivo studies are needed to understand the immune response to the graft. Health sciences/Endocrinology/Endocrine system and metabolic diseases/Diabetes/Type 1 diabetes mellitus Physical sciences/Engineering/Biomedical engineering Biological sciences/Biotechnology/Biomaterials/Implants Biological sciences/Biotechnology/Biomaterials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Diabetes mellitus is a multisystem endocrine disorder that occurs due to autoimmune destruction of insulin-producing pancreatic beta cells in individuals with type 1 diabetes (T1DM) or resistance to chronically elevated insulin levels in individuals with type 2 diabetes (T2DM). 1, 2 Chronic hyperglycemia caused by poorly controlled diabetes often results in severe damage to multiple organs, including renal failure, blindness, painful neuropathy, impaired wound healing, limb loss, and life-threatening infections. 3 Exogenous insulin therapy is the mainstay of treatment for both T1DM and T2DM, but exogenous insulin therapy involves frequent blood glucose checks, self-administered insulin injections, and a dependence on patient adherence. 4, 5 Pancreatic transplantation of human islet cells has been performed to restore endogenous insulin production, but there has been limited clinical success. Despite decades of research, islet cell transplantation has had limited success in achieving long-term freedom from exogenous insulin therapy for insulin-dependent type 1 diabetic patients. Currently, islet cell transplants are performed via the infusion of islet cells into the recipient's portal vein, and approximately 60% of transplanted cells die within three days of transplantation due to ischemic injury. 4 Additionally, significant immunosuppression is needed to avoid rejection, but these medications are also toxic to islet cells, resulting in additional insult. 5, 6 The use of immunosuppressants remains a significant barrier to the more widespread use of islet cell transplantation. Despite these challenges, there have been continued efforts to make this therapy clinically successful, as the ability to restore normoglycemia without invasive insulin injections or immunosuppression could revolutionize the treatment of over seven million insulin-dependent diabetic patients living in the United States. 6 To overcome the limitations of immunosuppression, significant efforts have been made to investigate the immunoisolation of islet cells with microencapsulation and microencapsulation devices to evade host immune defenses and eliminate the need for immunosuppression. The immunoisolation of islet cells was first described by Chick et al. in the 1970s. 7 In the 1990s, Monaco developed an islet cell containing a vascular arteriovenous shunt with a semipermeable hollow fiber membrane that was designed to be anastomosed to the iliac vasculature. 8 Despite significant efforts to create immunoisolating islet cell devices, including some devices used in clinical trials, there has been limited clinical success in freeing patients from exogenous insulin. 4, 7, 8, 9, 10, 11, 12, 13, 4 Limitations have been attributed primarily to vascular graft thrombosis and fibrosis. Additionally, all of these devices were transplanted outside of the anatomic location for islet cell insulin delivery, the portal vein, which is the site of delivery for traditional islet cell transplantation. We hypothesize that an islet cell-containing endovascular device designed for delivery to the portal circulation protects islets from the host immune system and promotes islet survival through free exchange of oxygen, glucose, and insulin with the host circulation. Successful immunoisolation drastically increases the access of T1DM patients to islet cell transplantation. The aim of this study was to outline the design and development of an immunoisolating islet cell vascular graft and demonstrate islet cell survival and function within the device via a benchtop model of mesenteric perfusion. Methods Animal care & monitoring Donor BALB/c mice were maintained in a specific-pathogen-free facility, in individually ventilated polycarbonate cages (Tecniplast GM500, 500 cm² floor area) on autoclaved hardwood-chip bedding, 4–5 animals per cage, 12 h light/12 h dark cycle (lights 07:00–19:00), temperature 22 ± 1 °C, relative humidity 50 ± 10 %, with ad libitum irradiated chow and water. Animals were inspected at least twice daily for the humane-endpoint criteria. Any animal meeting an endpoint would be humanely euthanized immediately. Islet Cell Procuring/Donor Pancreatectomy : All animal work was performed with the approval and oversight of the Massachusetts General Hospital Institutional Animal Care and Use Committee. Islet cells were isolated from 6–8-week-old male BALB/c mice (Jackson Laboratories, Bar Harbor, ME). Mice were anesthetized with inhaled isoflurane delivered in 100 % O₂ (induction 4 % at 1 L min⁻¹; maintenance 1.5–2 % at 0.5 L min⁻¹) until a surgical plane was reached, in accordance with IACUC Protocol. The mice were then euthanized by cervical dislocation. The abdomen was prepped with the animal in dorsal recumbency, and the abdominal wall was opened with a large flap using scissors. The flap was retracted caudally to expose the abdominal contents. The intestines were moved to the left side of the animal and traced to the duodenum to expose the gallbladder and common bile duct. Under magnification, the common bile duct was clamped distal to the confluence of the pancreatic duct. Using a 30 g needle, the common bile duct was cannulated proximally, and 2.5 cc of 1x Liberase (Sigma Aldrich, Burlington, MA) solution was infused retrograde through the pancreatic duct. The pancreas was then dissected from the surrounding tissue, and the pancreatic duct was transected. The excised pancreases were placed in 20 cc of 1x Liberase. All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition) and institutional regulations, and the study is reported in accordance with the ARRIVE 2.0 guidelines. Islet isolation and transplantation The murine pancreas was enzymatically digested with 1x Liberase for 15 minutes at 37°C. The islets were purified from the digestion mixture via a continuous gradient solution (densities of 1.11 to 1.06). Samples taken from different fractions after purification were used to assess the purity of each cell fraction. All pancreata meeting >50 % islet purity and viability were included; none were excluded. The final islet preparations were enumerated via manual counting, sizing, and conversion of the islet particle number (IPN) to islet equivalents (IEQ) on the basis of a 150 mm diameter. The remaining cells were incubated for 24 hours in low-glucose 2.8 mM cell culture media overnight. Rested cells were subjected to two washes with zero-glucose media and were prepared for injection in a 2 cc syringe via a 30 g needle. The device was seeded with 5000–6000 murine islet equivalents (IEQ). Human Islet Preparation Standard human islet isolation protocol (10,000 IEQ). Human islets were prepared by the MGH Center for Transplantation Sciences Islet Cell Core per the institutional protocol. IsletStent device The IsletStent consists of two concentric layers (Figure 1). 14 The outer component is an uncovered self-expanding stent designed for apposition to the portal vein wall without covering side-branch vessels. The outer layer is connected to an inner component through support struts. The inner component consists of an immuno-isolating cellular chamber. The semipermeable membrane for the cell chamber is made from porous (0.22 µm pore) ePTFE due to its favorable biocompatibility, suitable permeability characteristics and widespread usage in the manufacturing of FDA-approved vascular devices. This device is patent pending. Each prototype fabricated for these experiments was designed with a resealable channel for injecting cells into the cell chamber. After the injection of islet cells into the device, the channel was sealed, and the device was placed in zero-glucose media and maintained at 37°C for transport to the perfusion circuit. Normothermic Machine Perfusion Circuit: The normothermic machine perfusion model is summarized in Figure 2 and consists of a peristaltic pump, a membrane oxygenator, a heat exchanger, and a chamber to simulate intravascular deployment of the graft. The perfusion media in the circuit is dependent on the stage of the subsequent experiment; however, in addition to variable glucose levels, all media consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% albumin and 1% penicillin‒streptomycin (Thermo Fisher Scientific, Waltham, MA). At each medium change, the circuit was drained and cycled with Lactated Ringers w/10% albumin (Thermo Fisher Scientific, Waltham, MA) to remove any residual perfusion media. The volume of media in the perfusion circuit was held constant at 40 cc for all conditions. (Figure 3) Perfusion glucose-stimulated insulin response assays The IsletStent devices were placed within the perfusion circuit as depicted in Figure 2. This protocol is based on previously published protocols for nonperfusion GSIS studies. After the IsletStent devices were cellularized with murine or human islets, they were placed in the perfusion circuit under low-glucose (2.8 mM) conditions and allowed to rest for 24 hours. The following day, the devices were exposed to different levels of glucose in the perfusion media, and insulin levels were drawn at predetermined time points after the start of each condition. Zero-glucose conditions were not associated with insulin supplementation. The low-glucose conditions contained 2.8 mM glucose, and the high-glucose conditions contained 28 mM glucose. Each experimental condition consisted of 3 hours of perfusion with samples drawn at 1 minute, 1 hour, 2 hours, and 3 hours after the start of perfusion. After 3 hours of perfusion, the media was removed from the circuit and replaced with lactated Ringer’s (Thermo Fisher Scientific, Waltham, MA) solution for 15 minutes before being replaced with new media. Devices are exposed to lower glucose conditions prior to higher glucose conditions to prevent cells from continuing to respond to high glucose conditions, as they acclimatize to low glucose conditions, which could falsely increase insulin secretion under low glucose conditions. After completion of day 2, the GSIS devices were returned to low-glucose perfusion media, and normothermic perfusion was continued overnight. The samples were collected, immediately frozen, and stored at -80°C. Insulin levels were measured in each sample via ani-mouse insulin ELISA (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Our primary outcome measure was mean insulin concentration (mIU ml⁻¹) after 3 hours of perfusion. Statistical comparisons of insulin levels were performed between groups using a two-tailed paired t-tests in with α = 0.05. Insulin secretion rate assays The IsletStent devices were placed within the perfusion circuit as depicted in Figure 1. The devices were then exposed to low-glucose (2.8 mM) perfusion media and high-glucose perfusion media for 3 hours at each timepoint. However, at each hour, all of the media in the circuit were collected for sampling; thus, each time point contained the total amount of glucose secreted by the device over each hour. Since the volume of perfusion media remained constant at 40 cc for all experimental conditions, the total amount of insulin secreted, in mIU/hr, can be determined. Statistical comparisons of the rate of insulin secretion were performed between groups via paired t tests. Results In the perfusion GSIS using murine islet cells, under glucose-free perfusion conditions, the insulin level after 1 min of perfusion was 28 ± 12 mIU/ml, the insulin level after 1 h of perfusion was 71 ± 14 mIU/ml, the insulin level after 2 h of perfusion was 80 ± 9 mIU/ml, and the insulin level after 3 h of perfusion was 83 ± 17 mIU/ml. Under low-glucose conditions, the insulin level after 1 min of perfusion was 9 ± 4 mIU/ml, the insulin level after 1 h of perfusion was 26 ± 9 mIU/ml, the insulin level after 2 h of perfusion was 50 ± 3 mIU/ml, and the insulin level after 3 h of perfusion was 82 ± 8 mIU/ml. Under high-glucose conditions, the insulin level after the insulin level after 1 min of perfusion was 13 ± 1 mIU/ml, the insulin level after 1 h of perfusion was 103 ± 5 mIU/ml, the insulin level after 2 h of perfusion was 134 ± 17 mIU/ml, and the insulin level after 3 h of perfusion was 145 ± 20 mIU/ml (Figure 4). There were significantly greater levels of insulin secretion after exposure to high-glucose media. After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 83 ± 8 mIU/ml, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 145 ± 20 mIU/ml (Figure 5). The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.02; α = 0.05). In the insulin secretion rate assay, the IsletStent released 3,866 ± 68 mIU/hr in glucose-free conditions. In low-glucose media, the IsletStent released 5344 mIU/hr of insulin. Under high-glucose conditions, IsletStent released 6795 ± 503 mIU/hr (Figure 6). The rate of insulin secretion was significantly greater under high-glucose conditions than under low-glucose conditions (6759 ± 503 mIU/h vs. 5344 ± 144 mIU/hr, p = 0.008, α = 0.05). For the perfusion of GSIS using human islet cells, after 1 hour of low glucose exposure (2.8 mM), the average insulin concentration was 245 ± 6 mIU/dL, and after 1 hour of high glucose exposure (28 mM), the average insulin concentration was 321 ± 32 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.019; α = 0.05). After 2 hours of low glucose exposure (2.8 mM), the average insulin concentration was 237 ± 4 mIU/dL, and after 2 hours of high glucose exposure (28 mM), the average insulin concentration was 322 ± 16 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.002; α = 0.05). After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 229 ± 13 mIU/dL, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 300 ± 17 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.014; α = 0.05). Day 1 insulin release results are summarized in Figure 7. Discussion The current method of islet cell transplantation involves intravascular injection of islet clusters through the portal vein into the hepatic parenchymal bed. Approximately 60% of transplanted cells die within 3 days of transplantation. 6, 7 The primary drivers affecting islet cell survival after transplantation have been shown to be ischemic injury and toxicity from necessary immunosuppressive medications. 15, 16 These medications also carry a host of negative side effects, and the risks of these side effects often do not outweigh the benefit of transplantation for many patients. In an effort to free patients from both insulin and immunosuppressant medications, significant efforts have been made to investigate the immunoisolation of islet cells with microencapsulation and microencapsulation devices to evade host immune defenses. Immunoisolation through cellular microencapsulation was first described by Chang in the 1960s, 17 and immunoisolation of islet cells was first described by Chick et al . in the 1970s. 7 Many experimental immunoisolated islet cell devices have since been described, 8, 11, 12 including those used in clinical trials. 11, 13 Most recently, Viactye, which was purchased by Vertex Pharmaceutical in 2022, has shown promising results in humans because of its Pec-Encap technology, which uses an islet cell immunoisolation device designed for subcutaneous housing with expanded polytetrafluoroethylene (ePTFE) as a semipermeable membrane. 18, 19 In the phase II open-label multicenter trial using the PEC-Encap device with Vertex’s VC-02 pancreatic endoderm-derived islet cells, 3 of 10 patients had undetectable baseline C-peptide levels, and three achieved measurable C-peptide levels after 6 months, which correlated with improved CGM measures and reduced insulin dosing. 20 Unfortunately, all the patients continued to require exogenous insulin therapy despite device implantation. Notably, these devices were all implanted subcutaneously, unlike traditional islet cell transplantation via infusion into the portal vein. One of the major challenges that has been described in immunoisolated devices is maintaining a high enough oxygen concentration for islet survival; thus, the subcutaneous location of delivery may not provide adequate oxygenation. 13, 4, 21 Other authors have reported on devices designed to address the oxygen shortage in subcutaneous implantations. For example, Beta-O2 technologies produce a subcutaneous device called the BetaAir bioartificial pancreas, which encapsulates islet cells in an alginate hydrogel that increases oxygen availability through a refillable implanted oxygen container. 13, 21 The results of their initial 4-person clinical trial revealed that the βAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C-peptide were observed, and there was no impact on metabolic control. 13 To address the shortcomings of subcutaneous implantation, such as poor oxygenation and vascularization, hybrid devices have been developed that house islet cells within a vascular graft. These devices are designed to prevent ischemia by connecting the graft directly to the recipient’s blood vessels, thus allowing for immediate blood flow to the cells. In the 1990s, Monaco et al . developed a vascular arteriovenous shunt housing islet cells within a semipermeable hollow fiber membrane that was designed to be anastomosed to the iliac vasculature. 8 These shunts were transplanted into pancreatectomized canines and significantly reduced exogenous insulin requirements but did not result in freedom from exogenous insulin. Graft thrombosis was cited as a major reason for graft failure; however, antiplatelets and anticoagulants were not used in the initial study. The authors reported improvement in graft thrombosis rates in subsequent studies with the addition of aspirin therapy. 9, 22 Notably, these experiments were performed before the invention of novel antiplatelet and anticoagulant medications. With modern direct oral anticoagulants (DOACs), as well as clopidogrel and ticagrelor, a wide variety of agents can be used to reduce the risk of thrombosis. Additionally, the IsletStent is designed to be compatible with the binding of antithrombotic agents to the membrane surface; specifically, heparin can be bound to the ePTFE membrane of the IsletStent, similar to other heparin-bound vascular grafts that are currently FDA approved. More recently, authors have attempted to create vascularized bio grafts for islet cell transplantation in a tubular vascular graft that uses a semipermeable hydrogel to provide immunoisolation, in a “biovascular pancreas”. 23 Interestingly, this device houses islet cells on the outer surface of the vascular graft such that it is not in direct contact with the blood and instead gains perfusion from microvascular invasion. When implanted as an aortic graft in a small animal model of T1DM, the graft successfully restored euglycemia in a glucose tolerance test 3 months after implantation; however, the results were unable to be replicated in a porcine animal model. 23 The failure in the porcine animal model was attributed to poor cellularization of the grafts using porcine islet cells, which are known to be challenging to isolate. 23 Importantly, the site for surgical implantation of the device was the animal’s aorta, and previous islet cell vascular grafts were implanted as iliac arterial-venous shunts; neither of these locations recapitulates the native path of insulin delivery. The IsletStent builds on these previously described islet cell vascular grafts in several ways. First, the device is designed for modern minimally invasive endovascular delivery, meaning that the device is connected directly to the patient’s blood vessels from within the inside of the blood vessel, thus eliminating the need for open surgery. Second, to our knowledge, the IsletStent is the only device designed for intravascular deployment into the portal venous circulation, which is the native location for insulin secretion from the pancreas. Intraportal infusion of islet cells has been used almost exclusively as a delivery method for clinical islet transplantation, and it is the only transplantation site for islet cell engraftment and sustained graft function. However, none of the previously reported islet cell transplantation devices have utilized the portal vein or liver bed as the site for device implantation. An important aspect of intraportal insulin delivery is that insulin has a first-pass effect on the liver, where it modulates hepatic metabolism, such as by downregulating gluconeogenesis in the setting of food ingestion to maintain euglycemia. Using modern minimally invasive techniques for vascular and interventional radiology, specifically those related to transhepatic portal shunt creation, we can deploy the IsletStent into the portal vein transhepatically or via direct percutaneous access of the splenic vein. This deployment will enable immunoisolated islet cell delivery to the native pancreatic circulation while housing the cells in an endovascular graft that provides immediate cellular perfusion and prevents ischemic cell injury associated with the embolization of islets into the portal vein. Aside from the challenges of ischemia and immunosuppression, islet cell transplantation has traditionally also been limited by a lack of availability of donor cells for transplantation. However, this is an area of significant innovation at the moment, with new sources of islet cells currently in clinical trials. 24 The IsletStent is well suited to capitalize on the new availability of cells for transplantation, as the device provides ischemic and immune protection regardless of the source of cells in the device. With a growing supply of islet cells becoming available, the ability to eliminate immunosuppressive medications will open the door for potentially treating all insulin-dependent diabetic patients with islet cell transplantation. This study has several limitations. First, the in vitro nature of this study limits our ability to extrapolate if the device will restore euglycemia in an animal model of diabetes, and it does not allow for evaluation of the immunoisolating characteristics of the device. However, the benchtop perfusion model did allow us to demonstrate that islet cells were able to survive and function within the device, indicating that the membrane was appropriately permeable to enable rapid diffusion of oxygen, glucose, and insulin. Despite this limitation, the steps reported herein facilitate replication and provide a transparent basis for subsequent in-vivo translation. Future preclinical studies will evaluate the device in an ex vivo model of liver perfusion, which will help us understand how the IsletStent affects liver metabolism. Additionally, future in-vivo studies are needed to evaluate the immunoisolating capabilities of the graft, and although immunoisolation was not tested in this experiment, there is a strong foundation of prior studies reporting successful immunoisolation of islet cells via semipermeable membranes with characteristics similar to those of the IsletStent. Conclusion Human and murine islet cells within immunoisolated semipermeable stents perfused ex vivo secrete insulin in response to glucose stimulation in a statistically significant fashion. These results demonstrate free diffusion of glucose and insulin across a semipermeable PTFE membrane small enough to exclude large immunoglobulins and mediators of cellular immunity. These findings represent the significant promise of a minimally invasive endovascular islet-cell stent transplantation device that is designed to eliminate the need for systemic immunosuppression, which may hold the potential to be superior to standard islet-cell transplantation and free patients from the burdens of exogenous insulin therapy. Declarations Data Availability Statement The data from the murine and human islet cell experiments are readily available on request and summarized in Figures 4-7. Please contact Dr. Varun Singh at [email protected] for all data requests. Funding Statement Funding for the experiments described in the manuscript has been provided by the MESH Incubator, Boston, MA, the Mass General Brigham Gene and Cell Therapy Institute, Boston, MA, and the Massachusetts General Hospital Department of Surgery, Division of Transplant Surgery, Boston, MA. Author Contribution Statement These author contributed equally: Varun Singh and Brandon Gaston. V.S. and B.G. conceptualized and wrote the manuscript main text. A.K, K.D., K.L, J.L, J.M., and M.S. contributed to the review and revision of the manuscript. J.M. and M.S. provided advice, iterative report, and served as supervisors. Competing Interests Statement Dr. Singh and Dr. Gaston are listed inventors on a patent pending application (#PCT/US23/71910) which describes the securement device evaluated by this manuscript. 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CRISPR Therapeutics and ViaCyte Announce Strategic Collaboration to Develop Gene-Edited Stem Cell-Derived Therapy for Diabetes , (2018). Ramzy, A. et al. Implanted pluripotent stem-cell-derived pancreatic endoderm cells secrete glucose-responsive C-peptide in patients with type 1 diabetes. Cell Stem Cell 28 , 2047-2061.e2045 (2021). https://doi.org:https://doi.org/10.1016/j.stem.2021.10.003 Goswami, D. et al. Design Considerations for Macroencapsulation Devices for Stem Cell Derived Islets for the Treatment of Type 1 Diabetes. Advanced Science 8 , 2100820 (2021). https://doi.org:https://doi.org/10.1002/advs.202100820 Sullivan, S. J. et al. Biohybrid artificial pancreas: long-term implantation studies in diabetic, pancreatectomized dogs. Science 252 , 718-721 (1991). https://doi.org:10.1126/science.2024124 Han, E. X. et al. Development of a Bioartificial Vascular Pancreas. J Tissue Eng 12 , 20417314211027714 (2021). https://doi.org:10.1177/20417314211027714 Butler, P. C. & Gale, E. A. Reversing type 1 diabetes with stem cell-derived islets: a step closer to the dream? J Clin Invest 132 (2022). https://doi.org:10.1172/jci158305 Additional Declarations Competing interest reported. Dr. Singh and Dr. Gaston are listed inventors on a patent pending application (#PCT/US23/71910) which describes the securement device evaluated by this manuscript. As the intellectual property was created as part of an employment role, the employing institution, Massachusetts General Hospital, holds full ownership and rights to the intellectual property, in accordance with local legislation. None of the other authors have competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6441612","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468752925,"identity":"050860f5-c98c-4529-9937-023d5673f106","order_by":0,"name":"Brandon T. Gaston","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School (Co-first author), Mass General Brigham","correspondingAuthor":false,"prefix":"","firstName":"Brandon","middleName":"T.","lastName":"Gaston","suffix":""},{"id":468752926,"identity":"b85029a4-0a9d-42d8-9e27-d58a1f7f5f9d","order_by":1,"name":"Varun Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYBACgwOMDQyMDTZyDAw8DMwwQQYGNoJa0oxJ0QIkGBsOJzYQrUWy/3Dbw687Dqev7V97gLmg5l5i/+zDGxg+lB3GrWVGYrux7Jn03G033iUwzzhWnDjjXFoB44xz+LQwtklLtlkDtZwxYOZhSzBmOMNjwMzbhlsLP/9BkBbmdDOwln8JxvIgLX/xaWFIbJP82OacYHa+B2R4gpwBSAsjHi1sEolt0oxtaYbbbvAYHObtS5AzPMNWcLDnXDpuLfzHn0n+bLORNzt/xvAxz7cEHrkzzBsf/CizxqkFBJh5QKREAsMBmMgBXEphgPEH2FcE1Y2CUTAKRsFIBQAJ/Fkszkj3VQAAAABJRU5ErkJggg==","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School (Co-first author), Mass General Brigham","correspondingAuthor":true,"prefix":"","firstName":"Varun","middleName":"","lastName":"Singh","suffix":""},{"id":468752927,"identity":"137b34ed-aa3d-4155-9d50-270052ca6cc3","order_by":2,"name":"Anil Kharga","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Anil","middleName":"","lastName":"Kharga","suffix":""},{"id":468752928,"identity":"5b7036c9-c902-4f52-816f-d527e1b5532d","order_by":3,"name":"Kevin Deng","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School (Co-first author), Mass General Brigham","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Deng","suffix":""},{"id":468752929,"identity":"d279c636-c69a-40a2-b5ae-93fe3d2ad09c","order_by":4,"name":"Kang Mi Lee","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School (Co-first author), Mass General Brigham","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"Mi","lastName":"Lee","suffix":""},{"id":468752930,"identity":"9af5b769-4694-46ff-88bd-8457b34c7d43","order_by":5,"name":"Ji Lei","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School (Co-first author), Mass General Brigham","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"","lastName":"Lei","suffix":""},{"id":468752931,"identity":"ab5ea2d3-1d15-4d32-817b-5451a69761ae","order_by":6,"name":"James Markmann","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Markmann","suffix":""},{"id":468752932,"identity":"37187017-e108-4b62-ba32-7d051ca6011a","order_by":7,"name":"Marc D. Succi","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School (Co-first author), Mass General Brigham","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"D.","lastName":"Succi","suffix":""}],"badges":[],"createdAt":"2025-04-14 01:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6441612/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6441612/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84313593,"identity":"27f37299-41de-44d8-8028-85e0243e7e97","added_by":"auto","created_at":"2025-06-10 12:59:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1651152,"visible":true,"origin":"","legend":"\u003cp\u003e3D model of IsletStent. The inner tubular component is hollow and houses islet cells between two layers of semipermeable membrane to maximize the surface area for diffusion. The uncovered outer stent allows vessel wall apposition while maintaining side vessel perfusion. The device can be recellularized or removed from either direction via a proprietary snare and catheter system.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/7e91e73480bf76702f1a516e.png"},{"id":84314022,"identity":"9987623e-3fb4-47ce-9e26-c0949983eb51","added_by":"auto","created_at":"2025-06-10 13:07:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":421009,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of normothermic machine perfusion circuit for modeling mesenteric vascular perfusion. \u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/33bc27d195c78e9b05c36f81.png"},{"id":84313596,"identity":"80dbe348-7ec1-43bf-8605-98ef2ffde247","added_by":"auto","created_at":"2025-06-10 12:59:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1441664,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup for IsletStent ex-vivo perfusion study\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/938766fce7625a2770792dd7.png"},{"id":84315125,"identity":"238b7c45-5ce2-471c-a6ec-e5547f3bef76","added_by":"auto","created_at":"2025-06-10 13:15:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":191538,"visible":true,"origin":"","legend":"\u003cp\u003eHourly insulin levels for perfusion glucose stimulated insulin secretion (GSIS) assay.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/23ebedfb57c44a32ce261a90.png"},{"id":84315124,"identity":"c58279e7-dbc1-4704-b9b5-3aa345a86cd2","added_by":"auto","created_at":"2025-06-10 13:15:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":232891,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of insulin levels after 3 hours at low\u003c/p\u003e\n\u003cp\u003eglucose conditions vs. 3 hours at high glucose conditions.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/13f78b56145719791758b823.png"},{"id":84313599,"identity":"5049268b-b8a6-4d01-b2b8-ce5e4420b117","added_by":"auto","created_at":"2025-06-10 12:59:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":149508,"visible":true,"origin":"","legend":"\u003cp\u003eDay 1 average GSIS release results from human islet cells\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/88d9b8cfcc6bb042566aa831.png"},{"id":84313597,"identity":"e63d02b3-0c26-4c85-a10b-1e1dae6833e6","added_by":"auto","created_at":"2025-06-10 12:59:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":534729,"visible":true,"origin":"","legend":"\u003cp\u003eDay 1 glucose simulated insulin release results from human islets at each timepoint\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/8e918498312c948b382d2855.png"},{"id":95221880,"identity":"904b521f-ae9e-4dca-b38f-d57f13faa08c","added_by":"auto","created_at":"2025-11-05 16:19:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7875718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6441612/v1/b2606d7f-456a-462d-b798-42aaeff76c1f.pdf"}],"financialInterests":"Competing interest reported. Dr. Singh and Dr. Gaston are listed inventors on a patent pending application (#PCT/US23/71910) which describes the securement device evaluated by this manuscript. As the intellectual property was created as part of an employment role, the employing institution, Massachusetts General Hospital, holds full ownership and rights to the intellectual property, in accordance with local legislation. \nNone of the other authors have competing interests.","formattedTitle":"Development of an Immunoisolated Endovascular Islet Cell Stent to Treat Type I Diabetes Mellitus ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetes mellitus is a multisystem endocrine disorder that occurs due to autoimmune destruction of insulin-producing pancreatic beta cells in individuals with type 1 diabetes (T1DM) or resistance to chronically elevated insulin levels in individuals with type 2 diabetes (T2DM). \u003csup\u003e1, 2\u003c/sup\u003e Chronic hyperglycemia caused by poorly controlled diabetes often results in severe damage to multiple organs, including renal failure, blindness, painful neuropathy, impaired wound healing, limb loss, and life-threatening infections.\u003csup\u003e\u0026nbsp;3\u0026nbsp;\u003c/sup\u003eExogenous insulin therapy is the mainstay of treatment for both T1DM and T2DM, but exogenous insulin therapy involves frequent blood glucose checks, self-administered insulin injections, and a dependence on patient adherence.\u003csup\u003e\u0026nbsp;4, 5\u003c/sup\u003e Pancreatic transplantation of human islet cells has been performed to restore endogenous insulin production, but there has been limited clinical success.\u003c/p\u003e\n\u003cp\u003eDespite decades of research, islet cell transplantation has had limited success in achieving long-term freedom from exogenous insulin therapy for insulin-dependent type 1 diabetic patients. Currently, islet cell transplants are performed via the infusion of islet cells into the recipient\u0026apos;s portal vein, and approximately 60% of transplanted cells die within three days of transplantation due to ischemic injury.\u003csup\u003e\u0026nbsp;4\u003c/sup\u003e Additionally, significant immunosuppression is needed to avoid rejection, but these medications are also toxic to islet cells, resulting in additional insult. \u003csup\u003e5, 6\u003c/sup\u003e The use of immunosuppressants remains a significant barrier to the more widespread use of islet cell transplantation. Despite these challenges, there have been continued efforts to make this therapy clinically successful, as the ability to restore normoglycemia without invasive insulin injections or immunosuppression could revolutionize the treatment of over seven million insulin-dependent diabetic patients living in the United States.\u003csup\u003e\u0026nbsp;6\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo overcome the limitations of immunosuppression, significant efforts have been made to investigate the immunoisolation of islet cells with microencapsulation and microencapsulation devices to evade host immune defenses and eliminate the need for immunosuppression. The immunoisolation of islet cells was first described by Chick et al. in the 1970s.\u003csup\u003e\u0026nbsp;7\u0026nbsp;\u003c/sup\u003eIn the 1990s, Monaco developed an islet cell containing a vascular arteriovenous shunt with a semipermeable hollow fiber membrane that was designed to be anastomosed to the iliac vasculature.\u003csup\u003e\u0026nbsp;8\u0026nbsp;\u003c/sup\u003eDespite significant efforts to create immunoisolating islet cell devices, including some devices used in clinical trials, there has been limited clinical success in freeing patients from exogenous insulin.\u003csup\u003e\u0026nbsp;4,\u0026nbsp;7, 8, 9, 10, 11, 12, 13, 4\u0026nbsp;\u003c/sup\u003eLimitations have been attributed primarily to vascular graft thrombosis and fibrosis. Additionally, all of these devices were transplanted outside of the anatomic location for islet cell insulin delivery, the portal vein, which is the site of delivery for traditional islet cell transplantation.\u003c/p\u003e\n\u003cp\u003eWe hypothesize that an islet cell-containing endovascular device designed for delivery to the portal circulation protects islets from the host immune system and promotes islet survival through free exchange of oxygen, glucose, and insulin with the host circulation. Successful immunoisolation drastically increases the access of T1DM patients to islet cell transplantation. The aim of this study was to outline the design and development of an immunoisolating islet cell vascular graft and demonstrate islet cell survival and function within the device via a benchtop model of mesenteric perfusion.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003e\u003cu\u003eAnimal care \u0026amp; monitoring\u0026nbsp;\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDonor BALB/c mice were maintained in a specific-pathogen-free facility, in individually ventilated polycarbonate cages (Tecniplast GM500, 500 cm\u0026sup2; floor area) on autoclaved hardwood-chip bedding, 4\u0026ndash;5 animals per cage, 12 h light/12 h dark cycle (lights 07:00\u0026ndash;19:00), temperature 22 \u0026plusmn; 1 \u0026deg;C, relative humidity 50 \u0026plusmn; 10 %, with ad libitum irradiated chow and water. \u0026nbsp;Animals were inspected at least twice daily for the humane-endpoint criteria. Any animal meeting an endpoint would be humanely euthanized immediately.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003e\u003cbr\u003e\u0026nbsp;Islet Cell Procuring/Donor Pancreatectomy\u003c/u\u003e:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll animal work was performed with the approval and oversight of the Massachusetts General Hospital Institutional Animal Care and Use Committee. Islet cells were isolated from 6\u0026ndash;8-week-old male BALB/c mice (Jackson Laboratories, Bar Harbor, ME).\u0026nbsp;Mice were anesthetized with inhaled isoflurane delivered in 100 % O₂ (induction 4 % at 1 L min⁻\u0026sup1;; maintenance 1.5\u0026ndash;2 % at 0.5 L min⁻\u0026sup1;) until a surgical plane was reached, in accordance with IACUC Protocol.\u0026nbsp;The mice were then euthanized by cervical dislocation.\u0026nbsp;The abdomen was prepped with the animal in dorsal recumbency, and the abdominal wall was opened with a large flap using scissors. The flap was retracted caudally to expose the abdominal contents. The intestines were moved to the left side of the animal and traced to the duodenum to expose the gallbladder and common bile duct. Under magnification, the common bile duct was clamped distal to the confluence of the pancreatic duct. Using a 30 g needle, the common bile duct was cannulated proximally, and 2.5 cc of 1x Liberase (Sigma Aldrich, Burlington, MA) solution was infused retrograde through the pancreatic duct. The pancreas was then dissected from the surrounding tissue, and the pancreatic duct was transected. The excised pancreases were placed in 20 cc of 1x Liberase. All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition) and institutional regulations, and the study is reported in accordance with the ARRIVE 2.0 guidelines.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003e\u003cu\u003eIslet isolation and transplantation\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe murine pancreas was enzymatically digested with 1x Liberase for 15 minutes at 37\u0026deg;C. The islets were purified from the digestion mixture via a continuous gradient solution (densities of 1.11 to 1.06). Samples taken from different fractions after purification were used to assess the purity of each cell fraction. All pancreata meeting \u0026gt;50 % islet purity and viability were included; none were excluded.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe final islet preparations were enumerated via manual counting, sizing, and conversion of the islet particle number (IPN) to islet equivalents (IEQ) on the basis of a 150 mm diameter. The remaining cells were incubated for 24 hours in low-glucose 2.8 mM cell culture media overnight. Rested cells were subjected to two washes with zero-glucose media and were prepared for injection in a 2 cc syringe via a 30 g needle. The device was seeded with 5000\u0026ndash;6000 murine islet equivalents (IEQ).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003e\u003cu\u003eHuman Islet Preparation\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStandard human islet isolation protocol (10,000 IEQ). Human islets were prepared by the MGH Center for Transplantation Sciences Islet Cell Core per the institutional protocol.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003e\u003cu\u003eIsletStent device\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe IsletStent consists of two concentric layers (Figure 1). \u003csup\u003e14\u003c/sup\u003e The outer component is an uncovered self-expanding stent designed for apposition to the portal vein wall without covering side-branch vessels. The outer layer is connected to an inner component through support struts. The inner component consists of an immuno-isolating cellular chamber. The semipermeable membrane for the cell chamber is made from porous (0.22 \u0026micro;m\u0026nbsp;pore) ePTFE due to its favorable biocompatibility, suitable permeability characteristics and widespread usage in the manufacturing of FDA-approved vascular devices. This device is patent pending.\u003c/p\u003e\n\u003cp\u003eEach prototype fabricated for these experiments was designed with a resealable channel for injecting cells into the cell chamber. After the injection of islet cells into the device, the channel was sealed, and the device was placed in zero-glucose media and maintained at 37\u0026deg;C for transport to the perfusion circuit.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eNormothermic Machine Perfusion Circuit:\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe normothermic machine perfusion model is summarized in Figure 2 and consists of a peristaltic pump, a membrane oxygenator, a heat exchanger, and a chamber to simulate intravascular deployment of the graft. The perfusion media in the circuit is dependent on the stage of the subsequent experiment; however, in addition to variable glucose levels, all media consisted of Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 5% albumin and 1% penicillin‒streptomycin (Thermo Fisher Scientific, Waltham, MA). At each medium change, the circuit was drained and cycled with Lactated Ringers w/10% albumin (Thermo Fisher Scientific, Waltham, MA) to remove any residual perfusion media. The volume of media in the perfusion circuit was held constant at 40 cc for all conditions. (Figure 3)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003ePerfusion\u0026nbsp;glucose-stimulated insulin response assays\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe IsletStent devices were placed within the perfusion circuit as depicted in Figure 2. This protocol is based on previously published protocols for\u0026nbsp;nonperfusion\u0026nbsp;GSIS studies. After\u0026nbsp;the IsletStent devices were cellularized with murine or human islets, they were placed in the perfusion circuit\u0026nbsp;under\u0026nbsp;low-glucose (2.8 mM) conditions and allowed to rest for 24 hours. The following day,\u0026nbsp;the devices were exposed to different levels of glucose in the perfusion media,\u0026nbsp;and insulin levels were drawn at predetermined time points after the start of each condition. Zero-glucose conditions were\u0026nbsp;not associated with\u0026nbsp;insulin supplementation.\u0026nbsp;The low-glucose conditions contained 2.8 mM glucose, and\u0026nbsp;the high-glucose conditions contained 28 mM glucose. Each experimental condition consisted of 3 hours of perfusion with samples drawn at 1 minute, 1 hour, 2 hours, and 3 hours after the start of perfusion. After 3 hours of perfusion,\u0026nbsp;the media was removed from the circuit and replaced with lactated\u0026nbsp;Ringer\u0026rsquo;s (Thermo Fisher Scientific, Waltham, MA)\u0026nbsp;solution for 15\u0026nbsp;minutes\u0026nbsp;before being replaced with new media. Devices\u0026nbsp;are\u0026nbsp;exposed to lower glucose conditions prior to higher glucose conditions to prevent cells from continuing to respond to high glucose conditions,\u0026nbsp;as they acclimatize to low glucose conditions,\u0026nbsp;which could falsely\u0026nbsp;increase\u0026nbsp;insulin secretion\u0026nbsp;under\u0026nbsp;low glucose conditions. After completion of day 2,\u0026nbsp;the GSIS devices were returned to low-glucose perfusion media,\u0026nbsp;and normothermic perfusion\u0026nbsp;was continued overnight.\u003c/p\u003e\n\u003cp\u003eThe samples\u0026nbsp;were collected, immediately frozen, and stored at -80\u0026deg;C. Insulin levels were measured in each sample\u0026nbsp;via\u0026nbsp;ani-mouse insulin ELISA (Invitrogen,\u0026nbsp;Thermo Fisher Scientific, Waltham, MA). Our primary outcome measure was mean insulin concentration (mIU ml⁻\u0026sup1;) after 3 hours of perfusion. \u0026nbsp;Statistical comparisons of insulin levels were performed between groups using a two-tailed paired t-tests in with \u0026alpha; = 0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eInsulin\u0026nbsp;secretion rate assays\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe IsletStent devices were placed within the perfusion circuit as depicted in Figure 1. The devices were then exposed to low-glucose (2.8 mM) perfusion media and high-glucose perfusion media for 3 hours at each timepoint. However, at each hour, all of the media in the circuit were collected for sampling; thus, each time point contained the total amount of glucose secreted by the device over each hour. Since the volume of perfusion media remained constant at 40 cc for all experimental conditions, the total amount of insulin secreted, in mIU/hr, can be determined. Statistical comparisons of the rate of insulin secretion were performed between groups via paired t tests.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn the perfusion GSIS using murine islet cells, under glucose-free perfusion conditions, the insulin level after 1 min of perfusion was 28 \u0026plusmn; 12 mIU/ml, the insulin level after 1 h of perfusion was 71 \u0026plusmn; 14 mIU/ml, the insulin level after 2 h of perfusion was 80 \u0026plusmn; 9 mIU/ml, and the insulin level after 3 h of perfusion was 83 \u0026plusmn; 17 mIU/ml. Under low-glucose conditions, the insulin level after 1 min of perfusion was 9 \u0026plusmn; 4 mIU/ml, the insulin level after 1 h of perfusion was 26 \u0026plusmn; 9 mIU/ml, the insulin level after 2 h of perfusion was 50 \u0026plusmn; 3 mIU/ml, and the insulin level after 3 h of perfusion was 82 \u0026plusmn; 8 mIU/ml. Under high-glucose conditions, the insulin level after the insulin level after 1 min of perfusion was 13 \u0026plusmn; 1 mIU/ml, the insulin level after 1 h of perfusion was 103 \u0026plusmn; 5 mIU/ml, the insulin level after 2 h of perfusion was 134 \u0026plusmn; 17 mIU/ml, and the insulin level after 3 h of perfusion was 145 \u0026plusmn; 20 mIU/ml (Figure 4).\u003c/p\u003e\n\u003cp\u003eThere were significantly greater levels of insulin secretion after exposure to high-glucose media. After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 83 \u0026plusmn; 8 mIU/ml, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 145 \u0026plusmn; 20 mIU/ml (Figure 5). The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.02; \u0026alpha; = 0.05).\u003c/p\u003e\n\u003cp\u003eIn the insulin secretion rate assay, the IsletStent released 3,866 \u0026plusmn; 68 mIU/hr in glucose-free conditions. In low-glucose media, the IsletStent released 5344 mIU/hr of insulin. Under high-glucose conditions, IsletStent released 6795 \u0026plusmn; 503 mIU/hr (Figure 6).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe rate of insulin secretion was significantly greater under high-glucose conditions than under low-glucose conditions (6759 \u0026plusmn; 503 mIU/h vs. 5344 \u0026plusmn; 144 mIU/hr, p = 0.008, \u0026alpha; = 0.05).\u003c/p\u003e\n\u003cp\u003eFor the perfusion of GSIS using human islet cells, after 1 hour of low glucose exposure (2.8 mM), the average insulin concentration was 245 \u0026plusmn; 6 mIU/dL, and after 1 hour of high glucose exposure (28 mM), the average insulin concentration was 321 \u0026plusmn; 32 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.019; \u0026alpha; = 0.05). After 2 hours of low glucose exposure (2.8 mM), the average insulin concentration was 237 \u0026plusmn; 4 mIU/dL, and after 2 hours of high glucose exposure (28 mM), the average insulin concentration was 322 \u0026plusmn; 16 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.002; \u0026alpha; = 0.05). After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 229 \u0026plusmn; 13 mIU/dL, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 300 \u0026plusmn; 17 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.014; \u0026alpha; = 0.05). Day 1 insulin release results are summarized in Figure 7.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eThe current method of islet cell transplantation involves intravascular injection of islet clusters through the portal vein into the hepatic parenchymal bed. Approximately 60% of transplanted cells die within 3 days of transplantation.\u003csup\u003e\u0026nbsp;6, 7\u003c/sup\u003e The primary drivers affecting islet cell survival after transplantation have been shown to be ischemic injury and toxicity from necessary immunosuppressive medications.\u003csup\u003e\u0026nbsp;15, 16\u003c/sup\u003e These medications also carry a host of negative side effects, and the risks of these side effects often do not outweigh the benefit of transplantation for many patients. In an effort to free patients from both insulin and immunosuppressant medications, significant efforts have been made to investigate the immunoisolation of islet cells with microencapsulation and microencapsulation devices to evade host immune defenses.\u003c/p\u003e\n\u003cp\u003eImmunoisolation through cellular microencapsulation was first described by Chang in the 1960s,\u003csup\u003e\u0026nbsp;17\u003c/sup\u003e and immunoisolation of islet cells was first described by Chick \u003cem\u003eet al\u003c/em\u003e. in the 1970s.\u003csup\u003e\u0026nbsp;7\u0026nbsp;\u003c/sup\u003eMany experimental immunoisolated islet cell devices have since been described,\u003csup\u003e\u0026nbsp;8, 11, 12\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/sup\u003eincluding those used in clinical trials.\u003csup\u003e\u0026nbsp;11,\u0026nbsp;13\u003c/sup\u003e Most recently, Viactye, which was purchased by Vertex Pharmaceutical in 2022, has shown promising results in humans because of its Pec-Encap technology, which\u0026nbsp;uses\u0026nbsp;an islet cell immunoisolation device designed for subcutaneous\u0026nbsp;housing with expanded polytetrafluoroethylene (ePTFE) as a semipermeable membrane. \u003csup\u003e18, 19\u003c/sup\u003e In the phase II open-label multicenter trial using the PEC-Encap device with Vertex\u0026rsquo;s VC-02 pancreatic endoderm-derived islet cells, 3 of 10 patients had undetectable baseline C-peptide levels, and three achieved measurable C-peptide levels after 6 months, which correlated with improved CGM measures and reduced insulin dosing. \u003csup\u003e20\u003c/sup\u003e Unfortunately, all the patients continued to require exogenous insulin therapy despite device implantation. Notably, these devices were all implanted subcutaneously, unlike traditional islet cell transplantation via infusion into the portal vein. One of the major challenges that has been described in immunoisolated devices is maintaining a high enough oxygen concentration for islet survival; thus, the subcutaneous location of delivery may not provide adequate oxygenation.\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e13, 4, 21\u003c/sup\u003e Other authors have reported on devices designed to address the oxygen shortage in subcutaneous implantations. For example, Beta-O2 technologies produce a subcutaneous device called the BetaAir bioartificial pancreas, which encapsulates islet cells in an alginate hydrogel that increases oxygen availability through a refillable implanted oxygen container.\u003csup\u003e\u0026nbsp;13, 21\u0026nbsp;\u003c/sup\u003eThe results of their initial 4-person clinical trial revealed that the \u0026beta;Air device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C-peptide were observed, and there was no impact on metabolic control.\u003csup\u003e\u0026nbsp;13\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo address the shortcomings of subcutaneous implantation, such as poor oxygenation and vascularization, hybrid devices have been developed that house islet cells within a vascular graft. These devices are designed to prevent ischemia by connecting the graft directly to the recipient\u0026rsquo;s blood vessels, thus allowing for immediate blood flow to the cells. In the 1990s, Monaco \u003cem\u003eet al\u003c/em\u003e. developed a vascular arteriovenous shunt housing islet cells within a semipermeable hollow fiber membrane that was designed to be anastomosed to the iliac vasculature.\u003csup\u003e\u0026nbsp;8\u003c/sup\u003e These shunts were transplanted into pancreatectomized canines and significantly reduced exogenous insulin requirements but did not result in freedom from exogenous insulin. Graft thrombosis was cited as a major reason for graft failure; however, antiplatelets and anticoagulants were not used in the initial study. The authors reported improvement in graft thrombosis rates in subsequent studies with the addition of aspirin therapy. \u003csup\u003e9, 22\u003c/sup\u003e Notably, these experiments were performed before the invention of novel antiplatelet and anticoagulant medications. With modern direct oral anticoagulants (DOACs), as well as clopidogrel and ticagrelor, a wide variety of agents can be used to reduce the risk of thrombosis. Additionally, the IsletStent is designed to be compatible with the binding of antithrombotic agents to the membrane surface; specifically, heparin can be bound to the ePTFE membrane of the IsletStent, similar to other heparin-bound vascular grafts that are currently FDA approved.\u003c/p\u003e\n\u003cp\u003eMore recently, authors have attempted to create vascularized bio grafts for islet cell transplantation in a tubular vascular graft that uses a semipermeable hydrogel to provide immunoisolation, in a \u0026ldquo;biovascular pancreas\u0026rdquo;.\u003csup\u003e\u0026nbsp;23\u003c/sup\u003e Interestingly, this device houses islet cells on the outer surface of the vascular graft such that it is not in direct contact with the blood and instead gains perfusion from microvascular invasion. When implanted as an aortic graft in a small animal model of T1DM, the graft successfully restored euglycemia in a glucose tolerance test 3 months after implantation; however, the results were unable to be replicated in a porcine animal model.\u003csup\u003e\u0026nbsp;23\u003c/sup\u003e The failure in the porcine animal model was attributed to poor cellularization of the grafts using porcine islet cells, which are known to be challenging to isolate.\u003csup\u003e\u0026nbsp;23\u003c/sup\u003e Importantly, the site for surgical implantation of the device was the animal\u0026rsquo;s aorta, and previous islet cell vascular grafts were implanted as iliac arterial-venous shunts; neither of these locations recapitulates the native path of insulin delivery.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The IsletStent builds on these previously described islet cell vascular grafts in several ways. First, the device is designed for modern minimally invasive endovascular delivery, meaning that the device is connected directly to the patient\u0026rsquo;s blood vessels from within the inside of the blood vessel, thus eliminating the need for open surgery. Second, to our knowledge, the IsletStent is the only device designed for intravascular deployment into the portal venous circulation, which is the native location for insulin secretion from the pancreas. Intraportal infusion of islet cells has been used almost exclusively as a delivery method for clinical islet transplantation, and it is the only transplantation site for islet cell engraftment and sustained graft function. However, none of the previously reported islet cell transplantation devices have utilized the portal vein or liver bed as the site for device implantation. An important aspect of intraportal insulin delivery is that insulin has a first-pass effect on the liver, where it modulates hepatic metabolism, such as by downregulating gluconeogenesis in the setting of food ingestion to maintain euglycemia. Using modern minimally invasive techniques for vascular and interventional radiology, specifically those related to transhepatic portal shunt creation, we can deploy the IsletStent into the portal vein transhepatically or via direct percutaneous access of the splenic vein. This deployment will enable immunoisolated islet cell delivery to the native pancreatic circulation while housing the cells in an endovascular graft that provides immediate cellular perfusion and prevents ischemic cell injury associated with the embolization of islets into the portal vein.\u003c/p\u003e\n\u003cp\u003eAside from the challenges of ischemia and immunosuppression, islet cell transplantation has traditionally also been limited by a lack of availability of donor cells for transplantation. However, this is an area of significant innovation at the moment, with new sources of islet cells currently in clinical trials.\u003csup\u003e\u0026nbsp;24\u0026nbsp;\u003c/sup\u003eThe IsletStent is well suited to capitalize on the new availability of cells for transplantation, as the device provides ischemic and immune protection regardless of the source of cells in the device. With a growing supply of islet cells becoming available, the ability to eliminate immunosuppressive medications will open the door for potentially treating all insulin-dependent diabetic patients with islet cell transplantation.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. First, the in vitro nature of this study limits our ability to extrapolate if the device will restore euglycemia in an animal model of diabetes, and it does not allow for evaluation of the immunoisolating characteristics of the device. However, the benchtop perfusion model did allow us to demonstrate that islet cells were able to survive and function within the device, indicating that the membrane was appropriately permeable to enable rapid diffusion of oxygen, glucose, and insulin. Despite this limitation, the steps reported herein facilitate replication and provide a transparent basis for subsequent in-vivo translation. Future preclinical studies will evaluate the device in an ex vivo model of liver perfusion, which will help us understand how the IsletStent affects liver metabolism. Additionally, future in-vivo studies are needed to evaluate the immunoisolating capabilities of the graft, and although immunoisolation was not tested in this experiment, there is a strong foundation of prior studies reporting successful immunoisolation of islet cells via semipermeable membranes with characteristics similar to those of the IsletStent.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHuman and murine islet cells within immunoisolated semipermeable stents perfused ex vivo secrete insulin in response to glucose stimulation in a statistically significant fashion. These results demonstrate free diffusion of glucose and insulin across a semipermeable PTFE membrane small enough to exclude large immunoglobulins and mediators of cellular immunity. These findings represent the significant promise of a minimally invasive endovascular islet-cell stent transplantation device that is designed to eliminate the need for systemic immunosuppression, which may hold the potential to be superior to standard islet-cell transplantation and free patients from the burdens of exogenous insulin therapy.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data from the murine and human islet cell experiments are readily available on request and summarized in Figures 4-7. Please contact Dr. Varun Singh at [email protected] for all data requests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for the experiments described in the manuscript has been provided by the MESH Incubator, Boston, MA, the Mass General Brigham Gene and Cell Therapy Institute, Boston, MA, and the Massachusetts General Hospital Department of Surgery, Division of Transplant Surgery, Boston, MA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese author contributed equally: Varun Singh and Brandon Gaston.\u003c/p\u003e\n\u003cp\u003eV.S. and B.G. conceptualized and wrote the manuscript main text. A.K, K.D., K.L, J.L, J.M., and M.S. contributed to the review and revision of the manuscript. J.M. and M.S. provided advice, iterative report, and served as supervisors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Singh and Dr. Gaston are listed inventors on a patent pending application (#PCT/US23/71910) which describes the securement device evaluated by this manuscript. As the intellectual property was created as part of an employment role, the employing institution, Massachusetts General Hospital, holds full ownership and rights to the intellectual property, in accordance with local legislation. The authors disclose no other financial or non-financial competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026Ouml;stenson, C.-G. 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J.\u003cem\u003e et al.\u003c/em\u003e Biohybrid artificial pancreas: long-term implantation studies in diabetic, pancreatectomized dogs. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e252\u003c/strong\u003e, 718-721 (1991). https://doi.org:10.1126/science.2024124\u003c/li\u003e\n\u003cli\u003eHan, E. X.\u003cem\u003e et al.\u003c/em\u003e Development of a Bioartificial Vascular Pancreas. \u003cem\u003eJ Tissue Eng\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 20417314211027714 (2021). https://doi.org:10.1177/20417314211027714\u003c/li\u003e\n\u003cli\u003eButler, P. C. \u0026amp; Gale, E. A. Reversing type 1 diabetes with stem cell-derived islets: a step closer to the dream? \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e132\u003c/strong\u003e (2022). https://doi.org:10.1172/jci158305\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6441612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6441612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIntroduction and Objectives:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eDespite recent advances in cell sources and immunosuppression protocols, islet cell transplantation has had limited clinical success in achieving long-term freedom from exogenous insulin therapy for individuals with type 1 diabetes. Currently, islet cell transplants are performed via the infusion of islet cells into the recipient's portal vein. Approximately 60% of transplanted cells die within 3 days of transplantation due to ischemic injury.\u003csup\u003e7\u003c/sup\u003e Significant immunosuppression is needed to avoid rejection but is also toxic to islet cells, and the use of immunosuppression remains a barrier to more widespread use of islet cell transplantation. To address these challenges, we developed an endovascular biologic stent-graft, IsletStent, which is designed to utilize semipermeable membranes to protect transplanted islets from immune cell attack and prevent ischemic injury through endovascular deployment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eSemipermeable cell chambers in stent-grafts were fabricated from ePTFE with a pore size of 0.22 µm. The cell chamber was seeded with islet cells harvested from 6–8-week-old male BALB/c mice (Jackson Laboratories, Bar Harbor, ME). Human islets were obtained courtesy of an institutional islet isolation GMP facility. A benchtop normothermic machine perfusion circuit was used to model intravascular deployment. Within the perfusion circuit, devices were exposed to varying glucose concentrations in the perfusion media, and samples were drawn for insulin analysis. Insulin levels were measured via ELISA.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eIn murine islet cells, there were significantly increased levels of insulin secretion after exposure to high-glucose media. After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 83 ± 8 mIU/ml, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 145 ± 20 mIU/ml. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.02; α = 0.05). The rate of insulin secretion was significantly greater under high-glucose conditions than under low-glucose conditions (6759 ± 503 mIU/h vs. 5344 ± 144 mIU/hr, p = 0.008, α = 0.05). After 3 hours of low glucose exposure (2.8 mM), the average insulin concentration was 229 ± 13 mIU/dL, and after 3 hours of high glucose exposure (28 mM), the average insulin concentration was 300 ± 17 mIU/dl. The insulin concentration under high-glucose conditions was significantly greater than the insulin concentration under low-glucose conditions (p = 0.014; α = 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eThe IsletStent is a novel biologic endovascular device that enables islet cell function and survival within a semipermeable cell chamber placed intravascularly. However, further in-vivo studies are needed to understand the immune response to the graft.\u003c/p\u003e","manuscriptTitle":"Development of an Immunoisolated Endovascular Islet Cell Stent to Treat Type I Diabetes Mellitus ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 12:59:34","doi":"10.21203/rs.3.rs-6441612/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9277e65-dc36-40af-ac1d-7fb13d8eb0dc","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49767366,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Diabetes/Type 1 diabetes mellitus"},{"id":49767367,"name":"Physical sciences/Engineering/Biomedical engineering"},{"id":49767368,"name":"Biological sciences/Biotechnology/Biomaterials/Implants"},{"id":49767369,"name":"Biological sciences/Biotechnology/Biomaterials"}],"tags":[],"updatedAt":"2025-11-03T15:08:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 12:59:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6441612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6441612","identity":"rs-6441612","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}