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Introduction: Conventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200 IE/cm2. To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen. Methods: Oxygenation of individual islets under three culture conditions, standard low-density, non-GP high density, and GP high density, were first modeled with finite element simulations. Porcine islets from 30 preparations were cultured for 2 days in devices with GP membrane bottoms or in paired cultures under conventional conditions. Islets were seeded at high density (HD, â¼4000 IE/cm2, as measured by DNA) in both GP and non-GP devices. Results: In simulations, individual islets under standard culture conditions and high density cultures on GP membranes were both well oxygenated whereas non-GP high density cultured islets were anoxic. Similarly, compared to the non-GP paired controls, islet viability and recovery were significantly increased in HD GP cultures. The diabetes reversal rate in nude diabetic mice was similar for HD GP devices and standard cultures but was minimal with non-GP HD cultures. Discussion: Culturing islets in GP devices allows for a 20-fold increase of islet surface area density, greatly simplifying the culture process while maintaining islet viability and metabolism.
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Cellular macroencapsulation devices, known as tissue engineered grafts (TEGs), enable the transplantation of allogeneic cells without the need for life-long systemic immunosuppression. Islet containing TEGs offer promise as a potential functional cure for type 1 diabetes. Previous research has indicated sustained functionality of implanted islets at high density in a TEG requires external supplementary oxygen delivery and an effective tool to monitor TEG oxygen levels. A proven oxygen-measurement approach employs a 19F oxygen probe molecule (a perfluorocarbon) implanted alongside therapeutic cells to enable oxygen- and temperature- dependent NMR relaxometry. Although the approach has proved effective, the clinical translation of 19F oxygen relaxometry for TEG monitoring will be limited by the current inaccessibility and high cost of MRI. Here, we report the development of an affordable, compact, and tabletop 19F NMR relaxometry system for monitoring TEG oxygenation. The system uses a 0.5 T Halbach magnet with a bore diameter (19 cm) capable of accommodating the human arm, a potential site of future TEG implantation. 19F NMR relaxometry was performed while controlling the temperature and oxygenation levels of a TEG using a custom-built perfusion setup. Despite the magnet's nonuniform field, a pulse sequence of broadband adiabatic full-passage pulses enabled accurate 19F longitudinal relaxation rate (R1) measurements in times as short as â¼2 min (R1 vs oxygen partial pressure and temperature (R2 > 0.98)). The estimated sensitivity of R1 to oxygen changes at 0.5 T was 1.62-fold larger than the sensitivity previously reported for 16.4 T. We conclude that TEG oxygenation monitoring with a compact, tabletop 19F NMR relaxometry system appears feasible.
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Fluorocarbonos , Imageamento por Ressonância Magnética , Humanos , Espectroscopia de Ressonância Magnética , Oxigênio , TemperaturaRESUMO
Replacement of insulin-producing pancreatic beta-cells by islet transplantation offers a functional cure for type-1 diabetes (T1D). We recently demonstrated that a clinical grade alginate micro-encapsulant incorporating the immune-repellent chemokine and pro-survival factor CXCL12 could protect and sustain the integrity and function of autologous islets in healthy non-human primates (NHPs) without systemic immune suppression. In this pilot study, we examined the impact of the CXCL12 micro encapsulant on the function and inflammatory and immune responses of xenogeneic islets transplanted into the omental tissue bilayer sac (OB; n = 4) and diabetic (n = 1) NHPs. Changes in the expression of cytokines after implantation were limited to 2-6-fold changes in blood, most of which did not persist over the first 4 weeks after implantation. Flow cytometry of PBMCs following transplantation showed minimal changes in IFNγ or TNFα expression on xenoantigen-specific CD4+ or CD8+ T cells compared to unstimulated cells, and these occurred mainly in the first 4 weeks. Microbeads were readily retrievable for assessment at day 90 and day 180 and at retrieval were without microscopic signs of degradation or foreign body responses (FBR). In vitro and immunohistochemistry studies of explanted microbeads indicated the presence of functional xenogeneic islets at day 30 post transplantation in all biopsied NHPs. These results from a small pilot study revealed that CXCL12-microencapsulated xenogeneic islets abrogate inflammatory and adaptive immune responses to the xenograft. This work paves the way toward future larger scale studies of the transplantation of alginate microbeads with CXCL12 and porcine or human stem cell-derived beta cells or allogeneic islets into diabetic NHPs without systemic immunosuppression.
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Diabetes Mellitus , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Alginatos , Quimiocina CXCL12 , Sobrevivência de Enxerto , Terapia de Imunossupressão/métodos , Transplante das Ilhotas Pancreáticas/métodos , Projetos Piloto , Primatas , Suínos , Transplante Heterólogo/métodosRESUMO
In Type 1 diabetes patients, even ultra-rapid acting insulins injected subcutaneously reach peak concentrations in 45 minutes or longer. The lag time between dosing and peak concentration, as well as intra- and inter-subject variability, render prandial glucose control and dose consistency difficult. We postulated that insulin absorption from subcutaneously implantable vascularizing microchambers would be significantly faster than conventional subcutaneous injection. Male athymic nude R. norvegicus rendered diabetic with streptozotocin were implanted with vascularizing microchambers (single chamber; 1.5 cm2 surface area per side; nominal volume, 22.5 µl). Plasma insulin was assayed after a single dose (1.5 U/kg) of diluted insulin human (Humulin®R U-100), injected subcutaneously or via microchamber. Microchambers were also implanted in additional animals and retrieved at intervals for histologic assessment of vascularity. Following conventional subcutaneous injection, the mean peak insulin concentration was 22.7 (SD 14.2) minutes. By contrast, when identical doses of insulin were injected via subcutaneous microchamber 28 days after implantation, the mean peak insulin time was shortened to 7.50 (SD 4.52) minutes. Peak insulin concentrations were similar by either route; however, inter-subject variability was reduced when insulin was administered via microchamber. Histologic examination of tissue surrounding microchambers showed mature vascularization on days 21 and 40 post-implantation. Implantable vascularizing microchambers of similar design may prove clinically useful for insulin dosing, either intermittently by needle, or continuously by pump including in "closed loop" systems, such as the artificial pancreas.
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Diabetes Mellitus Tipo 1 , Insulina , Humanos , Masculino , Animais , Ratos , Camundongos , Insulina Regular Humana , Insulina Isófana , Camundongos NusRESUMO
Transplants comprised of encapsulated islets have shown promise in treating insulin-dependent diabetes. A question raised in the scientific and clinical communities is whether the insulin released from an implanted encapsulation device damaged in an accident could cause a serious hypoglycemic event. In this commentary, we consider the different types of damage that a device can sustain, including the encapsulation membrane and the islets within, and the amount of insulin released in each case. We conclude that the probability that device damage would cause an adverse hypoglycemic event is indeed very low.
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Diabetes Mellitus , Hipoglicemia , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Humanos , Transplante das Ilhotas Pancreáticas/efeitos adversos , Insulina , Hipoglicemiantes , Hipoglicemia/etiologia , Hipoglicemia/terapiaRESUMO
Introduction: Subcutaneous macroencapsulation devices circumvent disadvantages of intraportal islet therapy. However, a curative dose of islets within reasonably sized devices requires dense cell packing. We measured internal PO2 of implanted devices, mathematically modeled oxygen availability within devices and tested the predictions with implanted devices containing densely packed human islets. Methods: Partial pressure of oxygen (PO2) within implanted empty devices was measured by noninvasive 19F-MRS. A mathematical model was constructed, predicting internal PO2, viability and functionality of densely packed islets as a function of external PO2. Finally, viability was measured by oxygen consumption rate (OCR) in day 7 explants loaded at various islet densities. Results: In empty devices, PO2 was 12â mmHg or lower, despite successful external vascularization. Devices loaded with human islets implanted for 7 days, then explanted and assessed by OCR confirmed trends proffered by the model but viability was substantially lower than predicted. Co-localization of insulin and caspase-3 immunostaining suggested that apoptosis contributed to loss of beta cells. Discussion: Measured PO2 within empty devices declined during the first few days post-transplant then modestly increased with neovascularization around the device. Viability of islets is inversely related to islet density within devices.
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The delivery of encapsulated islets or stem cell-derived insulin-producing cells (i.e., bioartificial pancreas devices) may achieve a functional cure for type 1 diabetes, but their efficacy is limited by mass transport constraints. Modeling such constraints is thus desirable, but previous efforts invoke simplifications which limit the utility of their insights. Herein, we present a computational platform for investigating the therapeutic capacity of generic and user-programmable bioartificial pancreas devices, which accounts for highly influential stochastic properties including the size distribution and random localization of the cells. We first apply the platform in a study which finds that endogenous islet size distribution variance significantly influences device potency. Then we pursue optimizations, determining ideal device structures and estimates of the curative cell dose. Finally, we propose a new, device-specific islet equivalence conversion table, and develop a surrogate machine learning model, hosted on a web application, to rapidly produce these coefficients for user-defined devices.
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Diabetes Mellitus Tipo 1 , Insulinas , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Diabetes Mellitus Tipo 1/terapia , Humanos , Insulina , PâncreasRESUMO
A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p < .0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response.
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Diabetes Mellitus , Transplante das Ilhotas Pancreáticas , Animais , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Xenoenxertos , Humanos , Terapia de Imunossupressão , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Inflamação/etiologia , Transplante das Ilhotas Pancreáticas/métodos , Macaca fascicularis , Suínos , Transplante Heterólogo/métodosRESUMO
The islets of Langerhans of the pancreas are the primary endocrine organ responsible for regulating whole body glucose homeostasis. The use of isolated primary islets for research development and training requires organ resection, careful digestion, and isolation of the islets from nonendocrine tissue. This process is time consuming, expensive, and requires substantial expertise. For these reasons, we sought to develop a more rapidly obtainable and consistent model system with characteristic islet morphology and function that could be employed to train personnel and better inform experiments prior to using isolated rodent and human islets. Immortalized ß cell lines reflect several aspects of primary ß cells, but cell propagation in monolayer cell culture limits their usefulness in several areas of research, which depend on islet morphology and/or functional assessment. In this manuscript, we describe the propagation and characterization of insulinoma pseudo-islets (IPIs) from a rat insulinoma cell line INS832/3. IPIs were generated with an average diameter of 200 µm, consistent with general islet morphology. The rates of oxygen consumption and mitochondrial oxidation-reduction changes in response to glucose and metabolic modulators were similar to isolated rat islets. In addition, the dynamic insulin secretory patterns of IPIs were similar to primary rat islets. Thus, INS832/3-derived IPIs provide a valuable and convenient model for accelerating islet and diabetes research.
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Diabetes Mellitus/metabolismo , Insulinoma/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Consumo de Oxigênio/fisiologiaRESUMO
Cell encapsulation represents a promising therapeutic strategy for many hormone-deficient diseases such as type 1 diabetes (T1D). However, adequate oxygenation of the encapsulated cells remains a challenge, especially in the poorly oxygenated subcutaneous site. Here, we present an encapsulation system that generates oxygen (O2) for the cells from their own waste product, carbon dioxide (CO2), in a self-regulated (i.e., "inverse breathing") way. We leveraged a gas-solid (CO2-lithium peroxide) reaction that was completely separated from the aqueous cellular environment by a gas permeable membrane. O2 measurements and imaging validated CO2-responsive O2 release, which improved cell survival in hypoxic conditions. Simulation-guided optimization yielded a device that restored normoglycemia of immunocompetent diabetic mice for over 3 months. Furthermore, functional islets were observed in scaled-up device implants in minipigs retrieved after 2 months. This inverse breathing device provides a potential system to support long-term cell function in the clinically attractive subcutaneous site.
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Composite tissue (CT) preservation is important to outcomes after replant or transplant. Since the first limb replant, the mainstay of preservation has been static cold storage with the amputated part being placed in moistened gauze over ice. Historically, the gold-standard in solid organ preservation has been static cold storage with specialized solution, but this has recently evolved in the last few decades to develop technologies such as machine perfusion and even persufflation. This review explores the impact of cooling and oxygenation on CT, summarizes the work done in the area of CT preservation, discusses lessons learned from our experience in solid organ preservation, and proposes future directions.
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Preservação de Órgãos , Preservação de Tecido , Criopreservação , Extremidades , Humanos , PerfusãoRESUMO
BACKGROUND: We have utilized a noninvasive technique for measuring the partial pressure of oxygen (pO2) in alginate microcapsules implanted intraperitoneally in healthy nonhuman primates (NHPs). Average pO2 is important for determining if a transplant site and capsules with certain passive diffusion characteristics can support the islet viability, metabolic activity, and dose necessary to reverse diabetes. METHODS: Perfluoro-15-crown-5-ether alginate capsules were infused intraperitoneally into 3 healthy NHPs. Peritoneal pO2 levels were measured on days 0 and 7 using fluorine-19 magnetic resonance relaxometry and a fiber-optic probe. Fluorine-19 MRI was used to determine the locations of capsules within the peritoneal space on days 0 and 7. Gross and histologic evaluations of the capsules were used to assess their biocompatibility postmortem. RESULTS: At day 0 immediately after infusion of capsules equilibrated to room air, capsules were concentrated near the infusion site, and the pO2 measurement using magnetic resonance relaxometry was 147 ± 9 mm Hg. On day 7 after capsules were dispersed throughout the peritoneal cavity, the pO2 level was 61 ± 11 mm Hg. Measurements using the fiber-optic oxygen sensor were 132 ± 7.5 mm Hg (day 0) and 89 ± 6.1 mm Hg (day 7). Perfluoro-15-crown-5-ether capsules retrieved on day 7 were intact and free-floating without host cell attachment, although the numbers of peritoneal CD20 B cells, CD4 and CD8 T cells, and CD14 macrophages increased consistent with a mild foreign body reaction. CONCLUSIONS: The peritoneal pO2 of normal NHPs is relatively low and we predict would decrease further when encapsulated islets are transplanted intraperitoneally.
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Alginatos/farmacologia , Diabetes Mellitus Experimental/cirurgia , Imagem por Ressonância Magnética de Flúor-19/métodos , Transplante das Ilhotas Pancreáticas/métodos , Consumo de Oxigênio/fisiologia , Oxigênio/metabolismo , Cavidade Peritoneal/cirurgia , Animais , Cápsulas , Diabetes Mellitus Experimental/metabolismo , Feminino , Sobrevivência de Enxerto , Macaca mulatta , Pressão ParcialRESUMO
Stresses encountered during human islet isolation lead to unavoidable ß-cell death after transplantation. This reduces the chance of insulin independence in chronic pancreatitis patients undergoing total pancreatectomy and islet autotransplantation. We tested whether harvesting islets in carbon monoxide-saturated solutions is safe and can enhance islet survival and insulin independence after total pancreatectomy and islet autotransplantation. Chronic pancreatitis patients who consented to the study were randomized into carbon monoxide (islets harvested in a carbon monoxide-saturated medium) or control (islets harvested in a normal medium) groups. Islet yield, viability, oxygen consumption rate, ß-cell death (measured by unmethylated insulin DNA), and serum cytokine levels were measured during the peri-transplantation period. Adverse events, metabolic phenotypes, and islet function were measured prior and at 6 months post-transplantation. No adverse events directly related to the infusion of carbon monoxide islets were observed. Carbon monoxide islets showed significantly higher viability before transplantation. Subjects receiving carbon monoxide islets had less ß-cell death, decreased CCL23, and increased CXCL12 levels at 1 or 3 days post transplantation compared with controls. Three in 10 (30%) of the carbon monoxide subjects and none of the control subjects were insulin independent. This pilot trial showed for the first time that harvesting human islets in carbon monoxide-saturated solutions is safe for total pancreatectomy and islet autotransplantation patients.
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Monóxido de Carbono , Transplante das Ilhotas Pancreáticas/métodos , Pancreatite Crônica/terapia , Adolescente , Adulto , Idoso , Quimiocina CXCL12/sangue , Quimiocinas CC/sangue , Citocinas/sangue , Metilação de DNA , Intolerância à Glucose , Teste de Tolerância a Glucose , Humanos , Insulina/química , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/cirurgia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Pessoa de Meia-Idade , Pancreatectomia , Pancreatite Crônica/sangue , Pancreatite Crônica/metabolismo , Pancreatite Crônica/cirurgia , Qualidade de Vida , Inquéritos e Questionários , Fatores de Tempo , Transplante Autólogo/métodosRESUMO
KEY POINTS: Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity. Although whole-body glucose clearance is normal, 1-month-old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation. Impaired glucose-stimulated insulin secretion in IUGR lambs is due to lower intra-islet insulin availability and not from glucose sensing. We investigated adrenergic receptor (ADR) ß2 desensitization by administering oral ADRß modifiers for the first month after birth to activate ADRß2 and antagonize ADRß1/3. In IUGR lambs ADRß2 activation increased whole-body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies. IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose-stimulated insulin secretion and insulin-stimulated glucose oxidation, providing new insights into potential mechanisms for this risk. ABSTRACT: Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated ß adrenergic receptor (ADRß) desensitization. Our objectives were to measure insulin-sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRß2 agonist and ADRß1/ß3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose-stimulated insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR-AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole-body GUR were not different from controls. Of importance, ADRß2 stimulation with ß1/ß3 inhibition increases both insulin sensitivity and whole-body glucose utilization in IUGR lambs. In IUGR and IUGR-AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates and ex vivo skeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR-AR skeletal muscle than in controls but GLUT1 was greater in IUGR-AR. ADRß2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR-AR lambs heart rates were greater, which was independent of cardiac ADRß1 activation. We conclude that targeted ADRß2 stimulation improved whole-body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch-up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes.
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Retardo do Crescimento Fetal/tratamento farmacológico , Resistência à Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Antagonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Antagonistas de Receptores Adrenérgicos beta 2/farmacocinética , Antagonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Animais , Atenolol/administração & dosagem , Atenolol/farmacologia , Atenolol/uso terapêutico , Células Cultivadas , Clembuterol/administração & dosagem , Clembuterol/farmacologia , Clembuterol/uso terapêutico , Feminino , Retardo do Crescimento Fetal/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Músculo Esquelético/metabolismo , OvinosRESUMO
OBJECTIVE: This post hoc analysis evaluates the association between the frequency of diabetic foot ulcer (DFU) debridement and the proportion of ulcers treated with active continuous diffusion of oxygen (CDO) that heal in a 12-week evaluation period. MATERIALS AND METHODS: There were 146 patients with DFUs (77% men; average age, 56.3 ± 12.4 years) enrolled in a double-blind, placebo-controlled, randomized study to receive either active CDO or an otherwise fully operational placebo device. Patients were followed for 12 weeks or until wound closure. All patients received identical offloading, dressings, and follow-up. Ulcer debridement was left to the discretion of the treating physician and recorded from physician self-report as a dichotomous variable. RESULTS: A significantly higher proportion (204%) of ulcers healed in the CDO group compared with the placebo (46.2% vs. 22.6%, respectively; P = .016). The relative performance of active CDO over placebo became greater when frequent debridement was used (51.2% vs. 21.3%, respectively; P = .006). CONCLUSIONS: A significantly greater percentage of healing was recorded in patients receiving active CDO therapy than those receiving a placebo device in addition to standard wound care with identical dressings, debridement recommendations, and offloading. The relative performance of CDO appears to increase with the use of frequent debridement.
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Desbridamento , Pé Diabético/terapia , Oxigênio/uso terapêutico , Cicatrização/fisiologia , Adulto , Idoso , Bandagens , Pé Diabético/fisiopatologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: We previously demonstrated that the incorporation of the chemokine CXCL12 into alginate microbeads supported long-term survival of microencapsulated auto-, allo-, and xenogeneic islets in murine models of diabetes without systemic immune suppression. The purpose of this study was to test whether CXCL12 could abrogate foreign body responses (FBRs) against alginate microbeads which were empty or contained autologous islets in healthy nonhuman primates (NHPs; n = 4). METHODS: Two NHPs received intraperitoneal implants of 400 000 alginate microbeads with or without CXCL12, and postimplantation immunological and histopathological changes were evaluated up to 6 months postimplantation. A similar evaluation of autologous islets in CXCL12-containing alginate microbeads was performed in NHPs (n = 2). RESULTS: CXCL12-containing alginate microbeads were associated with a markedly reduced FBR to microbeads. Host responses to microbead implants were minimal, as assessed by clinical observations, blood counts, and chemistry. Evaluation of encapsulated islets was limited by the development of necrotizing pancreatitis after hemipancreatectomy in 1 NHP. A limited number of functioning islets were detectable at 6 months posttransplantation in the second NHP. In general, empty microbeads or islet-containing beads were found to be evenly distributed through the intraperitoneal cavity and did not accumulate in the Pouch of Douglas. CONCLUSIONS: Inclusion of CXCL12 in alginate microbeads minimized localized FBR. The NHP autologous islet implant model had limited utility for excluding inflammatory/immune responses to implanted islets because of the complexity of pancreatic surgery (hemipancreatectomy) before transplantation and the need to microencapsulate and transplant encapsulated autologous islets immediately after pancreatectomy and islet isolation.
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Human allogeneic islet transplantation (ITx) is emerging as a promising treatment option for qualified patients with type 1 diabetes. However, widespread clinical application of allogeneic ITx is hindered by two critical barriers: the need for systemic immunosuppression and the limited supply of human islet tissue. Biocompatible, retrievable immunoisolation devices containing glucose-responsive insulin-secreting tissue may address both critical barriers by enabling the more effective and efficient use of allogeneic islets without immunosuppression in the near-term, and ultimately the use of a cell source with a virtually unlimited supply, such as human stem cell-derived ß-cells or xenogeneic (porcine) islets with minimal or no immunosuppression. However, even though encapsulation methods have been developed and immunoprotection has been successfully tested in small and large animal models and to a limited extent in proof-of-concept clinical studies, the effective use of encapsulation approaches to convincingly and consistently treat diabetes in humans has yet to be demonstrated. There is increasing consensus that inadequate oxygen supply is a major factor limiting their clinical translation and routine implementation. Poor oxygenation negatively affects cell viability and ß-cell function, and the problem is exacerbated with the high-density seeding required for reasonably-sized clinical encapsulation devices. Approaches for enhanced oxygen delivery to encapsulated tissues in implantable devices are therefore being actively developed and tested. This review summarizes fundamental aspects of islet microarchitecture and ß-cell physiology as well as encapsulation approaches highlighting the need for adequate oxygenation; it also evaluates existing and emerging approaches for enhanced oxygen delivery to encapsulation devices, particularly with the advent of ß-cell sources from stem cells that may enable the large-scale application of this approach.
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Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/transplante , Transplante das Ilhotas Pancreáticas , Oxigênio , Animais , Hipóxia Celular , HumanosRESUMO
Diabetes mellitus (DM) is a common spontaneous endocrine disorder in dogs, which is defined by persistent hyperglycemia and insulin deficiency. Like type 1 diabetes (T1D) in people, canine DM is a complex and multifactorial disease in which genomic and epigenomic factors interact with environmental cues to induce pancreatic ß-cell loss and insulin deficiency, although the pathogenesis of canine DM is poorly defined and the role of autoimmunity is further controversial. Both diseases are incurable and require life-long exogenous insulin therapy to maintain glucose homeostasis. Human pancreatic islet physiology, size, and cellular composition is further mirrored by canine islets. Although pancreatic or isolated islets transplantation are the only clinically validated methods to achieve long-term normoglycemia and insulin independence, their availability does not meet the clinical need; they target a small portion of patients and have significant potential adverse effects. Therefore, providing a new source for ß-cell replacement is an unmet need. Naturally occurring DM in pet dogs, as a translational platform, is an untapped resource for various regenerative medicine applications that may offer some unique advantages given dogs' large size, longevity, heterogenic genetic background, similarity to human physiology and pathology, and long-term clinical management. In this review, we outline different strategies for curative approaches, animal models used, and consider the value of canine DM as a translational animal/disease model for T1D in people. Stem Cells Translational Medicine 2019;8:450-455.
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Diabetes Mellitus Experimental/terapia , Medicina Regenerativa/métodos , Animais , Modelos Animais de Doenças , Cães , HumanosRESUMO
BACKGROUND: All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. METHODS: Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of ß-cell function and RNA sequencing to elucidate transcriptomic changes. RESULTS: Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. CONCLUSIONS: These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved ß cell function.
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Temperatura Baixa , Mediadores da Inflamação/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Preservação de Órgãos/métodos , Oxigênio/farmacologia , Perfusão/métodos , Adolescente , Adulto , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Pessoa de Meia-Idade , Preservação de Órgãos/efeitos adversos , Via Secretória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Coleta de Tecidos e Órgãos , Adulto JovemRESUMO
Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) (GLP-1/Glb) for signaling efficacy in ß-cells. Expression of receptors for these ligands, as a combination, is relatively specific to the ß-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca2+, although Ca2+ responses were significantly depressed compared with the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at nonstimulatory glucose concentrations in INS 832/13 ß-cells or human islets of Langerhans. These data suggest that linking two binding elements, such as GLP-1 and Glb, into a single bivalent ligand can provide a unique functional agent targeted to ß-cells.