Bioactivation of Encapsulation Membranes Reduces Fibrosis and Enhances Cell Survival.

Encapsulation devices are an emerging barrier technology designed to prevent the immunorejection of replacement cells in regenerative therapies for intractable diseases. However, traditional polymers used in current devices are poor substrates for cell attachment and induce fibrosis upon implantation, impacting long-term therapeutic cell viability. Bioactivation of polymer surfaces improves local host responses to materials, and here we make the first step toward demonstrating the utility of this approach to improve cell survival within encapsulation implants. Using therapeutic islet cells as an exemplar cell therapy, we show that internal surface coatings improve islet cell attachment and viability, while distinct external coatings modulate local foreign body responses. Using plasma surface functionalization (plasma immersion ion implantation (PIII)), we employ hollow fiber semiporous poly(ether sulfone) (PES) encapsulation membranes and coat the internal surfaces with the extracellular matrix protein fibronectin (FN) to enhance islet cell attachment. Separately, the external fiber surface is coated with the anti-inflammatory cytokine interleukin-4 (IL-4) to polarize local macrophages to an M2 (anti-inflammatory) phenotype, muting the fibrotic response. To demonstrate the power of our approach, bioluminescent murine islet cells were loaded into dual FN/IL-4-coated fibers and evaluated in a mouse back model for 14 days. Dual FN/IL-4 fibers showed striking reductions in immune cell accumulation and elevated levels of the M2 macrophage phenotype, consistent with the suppression of fibrotic encapsulation and enhanced angiogenesis. These changes led to markedly enhanced islet cell survival and importantly to functional integration of the implant with the host vasculature. Dual FN/IL-4 surface coatings drive multifaceted improvements in islet cell survival and function, with significant implications for improving clinical translation of therapeutic cell-containing macroencapsulation implants.

Preferences of Type 1 Diabetic Patients on Devices for Islet Transplantation.

Transplantation of pancreatic islets within a biomaterial device is currently under investigation in clinical trials for the treatment of patients with type 1 diabetes (T1D). Patients' preferences on such implants could guide the designs of next-generation implantable devices; however, such information is not currently available. We surveyed the preferences of 482 patients with T1D on the size, shape, visibility, and transplantation site of islet containing implants. More than 83% of participants were willing to receive autologous stem cells, and there was no significant association between implant fabricated by one's own stem cell with gender (χ2 (1, n = 468) = 0.28; P = 0.6) or with age (χ2 (4, n = 468) = 2.92; P = 0.6). Preferred location for islet transplantation within devices was under the skin (52.7%). 48.3% preferred microscopic disks, and 32.3% preferred a thin device (like a credit card). Moreover, 58.4% preferred the implant to be as small as possible, 25.4% did not care about visibility, and 16.2% preferred their implants not to be visible. Among female participants, 81% cared about the implant visibility, whereas this number was 64% for male respondents (χ2 test (1, n = 468) = 16.34; P < 0.0001). 22% of those younger than 50 years of age and 30% of those older than 50 did not care about the visibility of implant (χ2 test (4, n = 468) = 23.69; P < 0.0001). These results suggest that subcutaneous sites and micron-sized devices are preferred choices among patients with T1D who participated in our survey.

Integration of Islet/Beta-Cell Transplants with Host Tissue Using Biomaterial Platforms.

Cell-based therapies are emerging for type I diabetes mellitus (T1D), an autoimmune disease characterized by the destruction of insulin-producing pancreatic ß-cells, as a means to provide long-term restoration of glycemic control. Biomaterial scaffolds provide an opportunity to enhance the manufacturing and transplantation of islets or stem cell-derived ß-cells. In contrast to encapsulation strategies that prevent host contact with the graft, recent approaches aim to integrate the transplant with the host to facilitate glucose sensing and insulin distribution, while also needing to modulate the immune response. Scaffolds can provide a supportive niche for cells either during the manufacturing process or following transplantation at extrahepatic sites. Scaffolds are being functionalized to deliver oxygen, angiogenic, anti-inflammatory, or trophic factors, and may facilitate cotransplantation of cells that can enhance engraftment or modulate immune responses. This local engineering of the transplant environment can complement systemic approaches for maximizing ß-cell function or modulating immune responses leading to rejection. This review discusses the various scaffold platforms and design parameters that have been identified for the manufacture of human pluripotent stem cell-derived ß-cells, and the transplantation of islets/ß-cells to maintain normal blood glucose levels.

A retrievable implant for the long-term encapsulation and survival of therapeutic xenogeneic cells.

The long-term function of transplanted therapeutic cells typically requires systemic immune suppression. Here, we show that a retrievable implant comprising a silicone reservoir and a porous polymeric membrane protects human cells encapsulated in it after implant transplantation in the intraperitoneal space of immunocompetent mice. Membranes with pores 1 µm in diameter allowed host macrophages to migrate into the device without the loss of transplanted cells, whereas membranes with pore sizes <0.8 µm prevented their infiltration by immune cells. A synthetic polymer coating prevented fibrosis and was necessary for the long-term function of the device. For >130 days, the device supported human cells engineered to secrete erythropoietin in immunocompetent mice, as well as transgenic human cells carrying an inducible gene circuit for the on-demand secretion of erythropoietin. Pancreatic islets from rats encapsulated in the device and implanted in diabetic mice restored normoglycaemia in the mice for over 75 days. The biocompatible device provides a retrievable solution for the transplantation of engineered cells in the absence of immunosuppression.

Transplantation of Islets of Langerhans into the Anterior Chamber of the Eye for Longitudinal In Vivo Imaging.

Noninvasive in vivo imaging techniques are attractive tools to longitudinally study various aspects of islet of Langerhans physiology and pathophysiology. Unfortunately, most imaging modalities currently applicable for clinical use do not allow the comprehensive investigation of islet cell biology due to limitations in resolution and/or sensitivity, while high-resolution imaging technologies like laser scanning microscopy (LSM) lack the penetration depth to assess islets of Langerhans within the pancreas. Significant progress in this area was made by the combination of LSM with the anterior chamber of the mouse eye platform, utilizing the cornea as a natural body window to study cell physiology of transplanted islets of Langerhans. We here describe the transplantation and longitudinal in vivo imaging of islets of Langerhans in the anterior chamber of the mouse eye as a versatile tool to study different features of islet physiology in health and disease.

VEGF-Modified PVA/Silicone Nanofibers Enhance Islet Function Transplanted in Subcutaneous Site Followed by Device-Less Procedure.

INTRODUCTION: As heterologous islets or islet-like stem cells become optional sources for islet transplantation, the subcutaneous site appears to be an acceptable replacement of the intrahepatic site due to its graft retrievability. The device-less (DL) procedure improves the feasibility; however, some limitations such as fibrotic overgrowth or immunodeficiency still exist. Nanofibers could mimic the extracellular matrix to improve the vitality of transplanted islets. Therefore, we designed a vascular endothelial growth factor (VEGF)-modified polyvinyl alcohol (PVA)/silicone nanofiber (SiO2-VEGF) to optimize the DL procedure. METHODS: SiO2-VEGF nanofibers were designed by nano-spinning and characterized the physical-chemical properties before subcutaneous islet transplantation. Cell viability, vessel formation, and glucose-stimulated insulin secretion were tested in vitro to ensure biocompatibility; and blood glucose level (BGL), transplanted islet function, and epithelial-mesenchymal transition (EMT)-related biomarker expression were analyzed in vivo. RESULTS: The intensity of inflammatory reaction induced by SiO2 nanofibers was between nylon and silicone, which did not bring out excessive fibrosis. The vascularization could be enhanced by VEGF functionalization both in vitro and in vivo. The BGL control was better in the DL combined with SiO2-VEGF group. The percentage of recipients that achieved normoglycemia was higher and earlier (71% at day 57), and the intraperitoneal glucose tolerance test (IPGTT) also confirmed better islet function. The expressions of vimentin, α-SMA, and twist-1 were upregulated, which indicated that SiO2-VEGF nanofibers might promote islet function by regulating the EMT pathway. DISCUSSION: In summary, our new SiO2-VEGF combined with DL procedure might improve the feasibility of subcutaneous islet transplantation for clinical application.

Evaluating Vascularization of Heterotopic Islet Constructs for Type 1 Diabetes Using an In Vitro Platform.

Type 1 diabetes (T1D) results from the autoimmune destruction of ß-cells within the pancreatic islets of Langerhans. Clinical islet transplantation from healthy donors is proposed to ameliorate symptoms, improve quality of life, and enhance the life span of afflicted T1D patients. However, post-transplant outcomes are dependent on the survival of the transplanted islets, which relies on the engraftment of the islets with the recipient's vasculature among other factors. Treatment strategies to improve engraftment include combining islets with supporting cells including endothelial cells (EC) and mesenchymal stem cells (MSC), dynamic cells capable of robust immunomodulatory and vasculogenic effects. In this study, we developed an in vitro model of transplantation to investigate the cellular mechanisms that enhance rapid vascularization of heterotopic islet constructs. Self-assembled vascular beds of fluorescently stained EC served as reproducible in vitro transplantation sites. Heterotopic islet constructs composed of islets, EC, and MSC were transferred to vascular beds for modeling transplantation. Time-lapsed imaging was performed for analysis of the vascular bed remodeling for parameters of neo-vascularization. Moreover, sampling of media following modeled transplantation showed secretory profiles that were correlated with imaging analyses as well as with islet function using glucose-stimulated insulin secretion. Together, evidence revealed that heterotopic constructs consisting of islets, EC, and MSC exhibited the most rapid recruitment and robust branching of cells from the vascular beds suggesting enhanced neo-vascularization compared to islets alone and control constructs. Together, this evidence supports a promising cell transplantation strategy for T1D and also demonstrates a valuable tool for rapidly investigating candidate cellular therapies for transplantation.

Engineering transferrable microvascular meshes for subcutaneous islet transplantation.

The success of engineered cell or tissue implants is dependent on vascular regeneration to meet adequate metabolic requirements. However, development of a broadly applicable strategy for stable and functional vascularization has remained challenging. We report here highly organized and resilient microvascular meshes fabricated through a controllable anchored self-assembly method. The microvascular meshes are scalable to centimeters, almost free of defects and transferrable to diverse substrates, ready for transplantation. They promote formation of functional blood vessels, with a density as high as ~220 vessels mm-2, in the poorly vascularized subcutaneous space of SCID-Beige mice. We further demonstrate the feasibility of fabricating microvascular meshes from human induced pluripotent stem cell-derived endothelial cells, opening a way to engineer patient-specific microvasculature. As a proof-of-concept for type 1 diabetes treatment, we combine microvascular meshes and subcutaneously transplanted rat islets and achieve correction of chemically induced diabetes in SCID-Beige mice for 3 months.

Cell Mass Increase Associated with Formation of Glucose-Controlling ß-Cell Mass in Device-Encapsulated Implants of hiPS-Derived Pancreatic Endoderm.

Device-encapsulated human stem cell-derived pancreatic endoderm (PE) can generate functional ß-cell implants in the subcutis of mice, which has led to the start of clinical studies in type 1 diabetes. Assessment of the formed functional ß-cell mass (FBM) and its correlation with in vivo metabolic markers can guide clinical translation. We recently reported ex vivo characteristics of device-encapsulated human embryonic stem cell-derived (hES)-PE implants in mice that had established a metabolically adequate FBM during 50-week follow-up. Cell suspensions from retrieved implants indicated a correlation with the number of formed ß cells and their maturation to a functional state comparable to human pancreatic ß cells. Variability in metabolic outcome was attributed to differences in number of PE-generated ß cells. This variability hinders studies on processes involved in FBM-formation. This study reports modifications that reduce variability. It is undertaken with device-encapsulated human induced pluripotent stem cell-derived-PE subcutaneously implanted in mice. Cell mass of each cell type was determined on intact tissue inside the device to obtain more precise data than following isolation and dispersion. Implants in a preformed pouch generated a glucose-controlling ß-cell mass within 20 weeks in over 60% of recipients versus less than 20% in the absence of a pouch, whether the same or threefold higher cell dose had been inserted. In situ analysis of implants indicated a role for pancreatic progenitor cell expansion and endocrine differentiation in achieving the size of ß- and α-cell mass that correlated with in vivo markers of metabolic control. Stem Cells Translational Medicine 2019;8:1296&1305.

Effect of Basic Fibroblast Growth Factor on Xenogeneic Islets in Subcutaneous Transplantation-A Murine Model.

BACKGROUND: Subcutaneous pockets provide an extrahepatic transplant site for islet grafting to treat type 1 diabetes. However, a hypoxic environment may cause central necrosis to islets and lead to graft failure. Our previous studies focused on a pre-treated subcutaneous site with basic fibroblast growth factor (bFGF) for the formation of vascular bed. In addition to neovascularization, bFGF was also shown to protect islets against oxidative stress and chemical-induced damage in vitro. Accordingly, we propose that subcutaneous islet transplantation with a bFGF-slow releasing device simultaneously can improve islet survival in vivo. METHODS: A bFGF-impregnated collagen sheet was implanted in the right back of a streptozotocin-induced diabetic mouse for neovascularization. After 10 days, the sheet was removed and the rat islet-embedding gel within the immune-isolation device was transplanted (2-time operation [OP]). In another group, the diabetic mice received bFGF-impregnated gel with rat islets within the immune-isolation device simultaneously (1-time OP). RESULTS: Diabetic mice in 2-time OP group experienced a decrease in their non-fasting blood glucose level for a period of 10 days, and the glucose levels were lower than those of untreated diabetic mice post-implantation. However, the mice in the 1-time OP group remained hyperglycemic post-operation and showed no improvements in body weight or the area under curve in intraperitoneal glucose tolerance test. Furthermore, mice in the 2-time OP had relatively higher serum insulin levels with improved renal and metabolic biomarkers. CONCLUSION: Our findings suggest that bFGF had no beneficial effect on a 1-time operation in subcutaneous islet transplantation.

Bioabsorbable device to prepare subcutaneous pockets for islet transplantation.

Small invasive transplantation of islets and long maintenance of the islet graft without immunosuppression has been studied for the treatment of type 1 diabetes. Clinically, subcutaneous pockets surrounding vascular-rich tissue are prepared for islet transplantation using a device made of the materials. Here, gelatin sheets were implanted into two dorsal subcutaneous sites in diabetic ACI rats, and a mixture of bFGF and sodium hyaluronate solution was injected around the gelatin sheets. A total of 1500 islets isolated from F344 rats were transplanted into each of the pockets 7 days after injection of the bFGF mixture. Nine of 10 diabetic ACI rats with allogeneic islet graft demonstrated long-term normoglycemia without administration of immunosuppressant. Gelatin sheets almost disappeared 67 days after implantation. Thus, subcutaneous immune-tolerant sites can be prepared using gelatin sheets and a sodium hyaluronate-bFGF mixture. Allogeneic islets transplanted into the sites can survive and control blood glucose levels for a long period, even without immunosuppression. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1107-1112, 2019.

Oxygen transporter for the hypoxic transplantation site.

Cell transplantation is a promising treatment for complementing lost function by replacing new cells with a desired function, e.g. pancreatic islet transplantation for diabetics. To prevent cell obliteration, oxygen supply is critical after transplantation, especially until the graft is sufficiently re-vascularized. To supply oxygen during this period, we developed a chemical-/electrical-free implantable oxygen transporter that delivers oxygen to the hypoxic graft site from ambient air by diffusion potential. This device is simply structured using a biocompatible silicone-based body that holds islets, connected to a tube that opens outside the body. In computational simulations, the oxygen transporter increased the oxygen level to >120 mmHg within grafts; in contrast, a control device that did not transport oxygen showed <6.5 mmHg. In vitro experiments demonstrated similar results. To test the effectiveness of the oxygen transporter in vivo, we transplanted pancreatic islets, which are susceptible to hypoxia, subcutaneously into diabetic rats. Islets transplanted using the oxygen transporter showed improved graft viability and cellular function over the control device. These results indicate that our oxygen transporter, which is safe and easily fabricated, effectively supplies oxygen locally. Such a device would be suitable for multiple clinical applications, including cell transplantations that require changing a hypoxic microenvironment into an oxygen-rich site.

An important step towards a prevascularized islet macroencapsulation device-effect of micropatterned membranes on development of endothelial cell network.

The development of immune protective islet encapsulation devices could allow for islet transplantation in the absence of immunosuppression. However, the immune protective membrane / barrier introduced there could also impose limitations in transport of oxygen and nutrients to the encapsulated cells resulting to limited islet viability. In the last years, it is well understood that achieving prevascularization of the device in vitro could facilitate its connection to the host vasculature after implantation, and therefore could provide sufficient blood supply and oxygenation to the encapsulated islets. However, the microvascular networks created in vitro need to mimic well the highly organized vasculature of the native tissue. In earlier study, we developed a functional macroencapsulation device consisting of two polyethersulfone/polyvinylpyrrolidone (PES/PVP) membranes, where a bottom microwell membrane provides good separation of encapsulated islets and the top flat membrane acts as a lid. In this work, we investigate the possibility of creating early microvascular networks on the lid of this device by combining novel membrane microfabrication with co-culture of human umbilical vein endothelial cell (HUVEC) and fibroblasts. We create thin porous microstructured PES/PVP membranes with solid and intermittent line-patterns and investigate the effect of cell alignment and cell interconnectivity as a first step towards the development of a stable prevascularized layer in vitro. Our results show that, in contrast to non-patterned membranes where HUVECs form unorganized HUVEC branch-like structures, for the micropatterned membranes, we can achieve cell alignment and the co-culture of HUVECs on a monolayer of fibroblasts attached on the membranes with intermittent line-pattern allows for the creation of HUVEC branch-like structures over the membrane surface. This important step towards creating early microvascular networks was achieved without the addition of hydrogels, often used in angiogenesis assays, as gels could block the pores of the membrane and limit the transport properties of the islet encapsulation device.

Immunoisolation of murine islet allografts in vascularized sites through conformal coating with polyethylene glycol.

Islet encapsulation may allow transplantation without immunosuppression, but thus far islets in large microcapsules transplanted in the peritoneal cavity have failed to reverse diabetes in humans. We showed that islet transplantation in confined well-vascularized sites like the epididymal fat pad (EFP) improved graft outcomes, but only conformal coated (CC) islets can be implanted in these sites in curative doses. Here, we showed that CC using polyethylene glycol (PEG) and alginate (ALG) was not immunoisolating because of its high permselectivity and strong allogeneic T cell responses. We refined the CC composition and explored PEG and islet-like extracellular matrix (Matrigel; MG) islet encapsulation (PEG MG) to improve capsule immunoisolation by decreasing its permselectivity and immunogenicity while allowing physiological islet function. Although the efficiency of diabetes reversal of allogeneic but not syngeneic CC islets was lower than that of naked islets, we showed that CC (PEG MG) islets from fully MHC-mismatched Balb/c mice supported long-term (>100 days) survival after transplantation into diabetic C57BL/6 recipients in the EFP site (750-1000 islet equivalents/mouse) in the absence of immunosuppression. Lack of immune cell penetration and T cell allogeneic priming was observed. These studies support the use of CC (PEG MG) for islet encapsulation and transplantation in clinically relevant sites without chronic immunosuppression.

Advances in polymeric islet cell encapsulation technologies to limit the foreign body response and provide immunoisolation.

Islet transplantation for the treatment of type 1 diabetes (T1D) is hampered by the shortage of donor tissue and the need for life-long immunosuppression. The engineering of materials to limit host immune rejection opens the possibilities of utilising allogeneic and even xenogeneic cells without the need for systemic immunosuppression. Here we discuss the most recent developments in immunoisolation of transplanted cells using advanced polymeric biomaterials, utilising macroscale to nanoscale approaches, to limit aberrant immune responses.

Local release of dexamethasone from macroporous scaffolds accelerates islet transplant engraftment by promotion of anti-inflammatory M2 macrophages.

Transplant-associated inflammatory responses generate an unfavorable microenvironment for tissue engraftment, particularly for cells susceptible to inflammatory stress, such as pancreatic islets. The localized delivery of anti-inflammatory agents, such as glucocorticoids, offers a promising approach to minimize the detrimental side effects associated with systemic delivery; however, the dosage must be carefully tailored to avoid deleterious responses, such as poor engraftment. Herein, we employed a polydimethylsiloxane (PDMS)-based three-dimensional scaffold platform for the local and controlled delivery of dexamethasone (Dex). Incorporation of 0.1% or 0.25% Dex within the scaffold was found to significantly accelerate islet engraftment in a diabetic mouse model, resulting in improved control of blood glucose levels during the early transplant period. Investigation into the mechanism of this impact found that local Dex delivery promotes macrophage polarization towards an anti-inflammatory (M2) phenotype and suppresses inflammatory pathways during the first week post-implantation. Alternatively, higher Dex loadings (0.5% and 1%) significantly delayed islet engraftment and function by impairing host cell migration into the implanted graft. Our results demonstrate the dose-dependent impact of local glucocorticoid delivery on the modulation of inflammatory responses at the implant site in vivo. Outcomes highlight the potential of this platform for generating favorable host responses that improve overall cellular transplant outcomes.

Bioengineering Strategies for the Treatment of Type I Diabetes.

Diabetes mellitus, the third most common disease in the world, is a chronic metabolic disorder caused by a failure of insulin production and/or an inability to respond to insulin. Specifically, type 1 diabetes is a disease resulted by the autoimmune destruction of a patient's ß-cell population within the pancreatic islets of Langerhans. The current primary treatment for type 1 diabetes is daily multiple insulin injections. However, this treatment cannot provide sustained physiological release, and the insulin amount is not finely tuned to glycemia. Pancreatic transplants or islet transplants would be the preferred treatment method but the lack of donor tissue and immunoincompatibility has been shown to be a roadblock to their widespread use. Bioengineering strategies are poised to combat these challenges. In this review, bioengineering approaches for the treatment of type 1 diabetes, including insulin controlled release systems, strategies for immunoisolation of transplanted islets, and cell-based therapies, such as ß-cells and stem cells, are discussed.

Mold-casted non-degradable, islet macro-encapsulating hydrogel devices for restoration of normoglycemia in diabetic mice.

Islet transplantation is a potential cure for diabetic patients, however this procedure is not widely adopted due to the high rate of graft failure. Islet encapsulation within hydrogels is employed to provide a three-dimensional microenvironment conducive to survival of transplanted islets to extend graft function. Herein, we present a novel macroencapsulation device, composed of PEG hydrogel, that combines encapsulation with lithography techniques to generate polydimethylsiloxane (PDMS) molds. PEG solutions are mixed with islets, which are then cast into PDMS molds for subsequent crosslinking. The molds can also be employed to provide complex architectures, such as microchannels that may allow vascular ingrowth through pre-defined regions of the hydrogel. PDMS molds allowed for the formation of stable gels with encapsulation of islets, and in complex architectures. Hydrogel devices with a thickness of 600 µm containing 500 islets promoted normoglycemia within 12 days following transplantation into the epididymal fat pad, which was sustained over the two-month period of study until removal of the device. The inclusion of microchannels, which had a similar minimum distance between islets and the hydrogel surface, similarly promoted normoglycemia. A glucose challenge test indicated hydrogel devices achieved normoglycemia 90 min post-dextrose injections, similar to control mice with native pancreata. Histochemical staining revealed that transplanted islets, identified as insulin positive, were viable and isolated from host tissue at 8 weeks post-transplantation, yet devices with microchannels had tissue and vascular ingrowth within the channels. Taken together, these results demonstrate a system for creating non-degradable hydrogels with complex geometries for encapsulating islets capable of restoring normoglycemia, which may expand islet transplantation as a treatment option for diabetic patients. Biotechnol. Bioeng. 2016;113: 2485-2495. © 2016 Wiley Periodicals, Inc.