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Introduction: Transplantation of kidneys from expanded criteria donors (ECD), including after circulatory death (DCD), is associated with a higher risk of adverse events compared to kidneys from standard criteria donors. In previous studies, improvements in renal transplant outcomes have been seen when kidneys were perfused with gaseous oxygen during preservation (persufflation, PSF). In the present study, we assessed ex-vivo renal function from a Diffusion Contrast Enhanced (DCE)-MRI estimation of glomerular filtration rate (eGFR); and metabolic sufficiency from whole-organ oxygen consumption (WOOCR) and lactate production rates., Methods: Using a porcine model of DCD, we assigned one kidney to antegrade PSF, and the contralateral kidney to static cold storage (SCS), both maintained for 24 h at 4°C. Post-preservation organ quality assessments, including eGFR, WOOCR and lactate production, were measured under cold perfusion conditions, and biopsies were subsequently taken for histopathological analysis., Results: A significantly higher eGFR (36.6 ± 12.1 vs. 11.8 ± 4.3 ml/min, p  < 0.05), WOOCR (182 ± 33 vs. 132 ± 21 nmol/min*g, p  < 0.05), and lower rates of lactate production were observed in persufflated kidneys. No overt morphological differences were observed between the two preservation methods., Conclusion: These data suggest that antegrade PSF is more effective in preserving renal function than conventional SCS. Further studies in large animal models of transplantation are required to investigate whether integration with PSF of WOOCR, eGFR or lactate production measurements before transplantation are predictive of post-transplantation renal function and clinical outcomes., Competing Interests: BW and MT formed Sylvatica Biotech, Inc., after the investigations reported herein were completed. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Min, Galons, Lynch, Steyn, Price, Weegman, Taylor, Pandey, Harland, Martin, Besselsen, Putnam and Papas.)

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/cm 2 . 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, ∼4000 IE/cm 2 , 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Avgoustiniatos, Mueller, Scott III, Kitzmann, Suszynski, Perrault, Falde, Balamurugan, Hering, Putnam and Papas.)

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 19 F 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 19 F 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 19 F 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. 19 F 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 19 F longitudinal relaxation rate (R 1 ) measurements in times as short as ∼2 min (R 1 vs oxygen partial pressure and temperature (R 2  > 0.98)). The estimated sensitivity of R 1 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 19 F NMR relaxometry system appears feasible., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Efrain Torres reports a relationship with Adialante LLC that includes: equity or stocks. Parker Jenkins reports a relationship with Adialante LLC that includes: equity or stocks. Dimitrios Sakellariou reports a relationship with RE Magnet Studio Ltd that includes: equity or stocks. Klearchos K. Papas reports a relationship with Procyon Technologies LLC that includes: equity or stocks., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 19 F-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 12 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., Competing Interests: KP is the co-founder and CEO of Procyon Technologies, LLC, a startup company focused on the development of oxygenated cell encapsulation devices. BW, who was a graduate student at the time of this study, is now employed by Sylvatica Biotech Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author MG declared that he was an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2023 Einstein, Steyn, Weegman, Suszynski, Sambanis, O'Brien, Avgoustiniatos, Firpo, Graham, Janecek, Eberly, Garwood, Putnam and Papas.)

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., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: [R.J. and KKP are co-founders of and have financial interests in Procyon Technologies LLC.]. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Steyn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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., (© 2022. The Author(s).)

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., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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 β cell lines reflect several aspects of primary β 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.

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 (O 2 ) for the cells from their own waste product, carbon dioxide (CO 2 ), in a self-regulated (i.e., "inverse breathing") way. We leveraged a gas-solid (CO 2 -lithium peroxide) reaction that was completely separated from the aqueous cellular environment by a gas permeable membrane. O 2 measurements and imaging validated CO 2 -responsive O 2 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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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.

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.

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) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 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 β adrenergic receptor (ADRβ) desensitization. Our objectives were to measure insulin-sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRβ2 agonist and ADRβ1/β3 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, ADRβ2 stimulation with β1/β3 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. ADRβ2, 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 ADRβ1 activation. We conclude that targeted ADRβ2 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., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Stresses encountered during human islet isolation lead to unavoidable β-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, β-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 β-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.

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.

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 β-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 β-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., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

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.

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 β-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 β-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 β-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 β-cell sources from stem cells that may enable the large-scale application of this approach., (Copyright © 2019. Published by Elsevier B.V.)

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 β-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 β cell function.

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 β-cells. Expression of receptors for these ligands, as a combination, is relatively specific to the β-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca 2+ , although Ca 2+ 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 β-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 β-cells.

Background: There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted., Methods: Neonatal porcine islets (NPI; 1-3 days), juvenile porcine islets (JPI; 18-21 days), and adult porcine islets (API; 2+ years) were compared in vitro, including assessments of oxygen consumption rate, membrane integrity determined by FDA/PI staining, β-cell proliferation, dynamic glucose-stimulated insulin secretion, and RNA sequencing., Results: Oxygen consumption rate normalized to DNA was not significantly different between ages. Membrane integrity was age dependent, and API had the highest percentage of intact cells. API also had the highest glucose-stimulated insulin secretion response during a dynamic insulin secretion assay and had 50-fold higher total insulin content compared to NPI and JPI. NPI and JPI had similar glucose responsiveness, β-cell percentage, and β-cell proliferation rate. Transcriptome analysis was consistent with physiological assessments. API transcriptomes were enriched for cellular metabolic and insulin secretory pathways, while NPI exhibited higher expression of genes associated with proliferation., Conclusions: The oxygen demand, membrane integrity, β-cell function and proliferation, and transcriptomes of islets from API, JPI, and NPI provide a comprehensive physiological comparison for future studies. These assessments will inform the optimal application of each age of porcine islet to expand the availability of islet transplantation., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Insulin secretion is stimulated by glucose metabolism and inhibited by catecholamines through adrenergic receptor stimulation. We determined whether catecholamines suppress oxidative metabolism in β-cells through adrenergic receptors. In Min6 cells and isolated rat islets, epinephrine decreased oxygen consumption rates compared to vehicle control or co-administration of epinephrine with α2-adrenergic receptor antagonist yohimbine. Epinephrine also decreased forskolin-stimulated oxygen consumption rates, indicating cAMP dependent and independent actions. Furthermore, glucose oxidation rates were decreased with epinephrine, independent of the exocytosis of insulin, which was blocked with yohimbine. We evaluated metabolic targets through proteomic analysis after 4 h epinephrine exposure that revealed 466 differentially expressed proteins that were significantly enriched for processes including oxidative metabolism, protein turnover, exocytosis, and cell proliferation. These results demonstrate that acute α2-adrenergic stimulation suppresses glucose oxidation in β-cells independent of nutrient availability and insulin exocytosis, while cAMP concentrations are elevated. Proteomics and immunoblots revealed changes in electron transport chain proteins that were correlated with lower metabolic reducing equivalents, intracellular ATP concentrations, and altered mitochondrial membrane potential implicating a new role for adrenergic control of mitochondrial function and ultimately insulin secretion., (Copyright © 2018 Elsevier B.V. All rights reserved.)

There is currently a significant disparity between the number of patients who need lifesaving transplants and the number of donated human organs. Xenotransplantation is a way to address this disparity and attempts to enable the use of xenogeneic tissues have persisted for centuries. While immunologic incompatibilities have presented a persistent impediment to their use, encapsulation may represent a way forward for the use of cell-based xenogeneic therapeutics without the need for immunosuppression. In conjunction with modern innovations such as the use of bioprinting, incorporation of immune modulating molecules into capsule membranes, and genetic engineering, the application of xenogeneic cells to treat disorders ranging from pain to liver failure is becoming increasingly realistic. The present review discusses encapsulation in the context of xenotransplantation, focusing on the current status of clinical trials, persistent issues such as antigen shedding, oxygen availability, and donor selection, and recent developments that may address these limitations., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: The aim of this study was to assess whether continuous diffusion of oxygen improves healing in people receiving treatment for diabetic foot ulcers (DFU)., Method: A double-blind, placebo control randomised study to receive either active continuous diffusion of oxygen (CDO) therapy using an active CDO device, or a fully operational placebo device without delivering oxygen. Patients were followed until closure or 12 weeks. Patients, caretakers, treating physicians and independent evaluators were blinded to the study arm. All patients received identical offloading, debridement, dressings and follow-up., Results: We enrolled 146 people with DFUs (77% male, aged 56.3±12.4 years). A significantly higher proportion (195%) of DFUs healed in the CDO arm compared with placebo (32.4% versus 16.7%, p=0.033). The time to 50% DFU closure was significantly shorter in patients that received CDO therapy (mean 18.4 versus 28.9 days, p=0.001). There were no differences in overall adverse events (p=0.66) or ulcer-related adverse events (p=0.30) in the active and placebo treatment groups. The relative performance of active CDO over placebo became greater when used in larger wounds (273%), in more chronic wounds (334%) and in weight bearing wounds (465%)., Conclusion: The results of this study demonstrate that CDO leads to higher proportion of healed DFUs (p=0.033) and a faster time to closure compared with placebo in people with DFUs (p=0.015). Relative performance did not vary significantly with wound size (p=0.80), but revealed better relative performance in more chronic wounds (p=0.008) and in weight-bearing wounds (p=0.003).

Purpose of Review: To summarize current literature and recent findings on the potential of humidified oxygenated gas perfusion (persufflation) as an alternative method for improved organ preservation., Recent Findings: Although there are some conflicting data, the majority of the evidence suggests that persufflation, by enhancing oxygenation, can improve preservation and even rescue organs, including organs with prior exposure to warm ischemia. In some cases, persufflation produced better results than hypothermic machine perfusion. The timing of persufflation is of importance; benefits of persufflation appear to increase as the timing of its administration postprocurement decreases. This may be particularly true for tissues that are more sensitive to ischemia, such as the pancreas prior to islet isolation. Combining oxygen persufflation with nitric oxide and addition of pulsatile flow may provide further benefits and amplify its effects on improving transplant outcomes., Summary: Persufflation is a promising, relatively simple, preservation technique that enables improved oxygenation, which provides protection and improvement in the graft condition during preservation and prior to transplantation. More detailed studies are needed to optimize persufflation and evaluate its short and long-term effects in vivo.

Background: Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived β cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention., Methods: Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression., Results: Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic β cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism., Conclusions: In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to β cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and β cell function and leads to increased inflammatory signaling.

Transplantation of macroencapsulated tissue-engineered grafts (TEGs) is being investigated as a treatment for type 1 diabetes, but there is a critical need to measure TEG viability both in vitro and in vivo. Oxygen deficiency is the most critical issue preventing widespread implementation of TEG transplantation and delivery of supplemental oxygen (DSO) has been shown to enhance TEG survival and function in vivo. In this study, we demonstrate the first use of oxygen-17 magnetic resonance spectroscopy ( 17 O-MRS) to measure the oxygen consumption rate (OCR) of TEGs and show that in addition to providing therapeutic benefits to TEGs, DSO with 17 O 2 can also enable measurements of TEG viability. Macroencapsulated TEGs containing βTC3 murine insulinoma cells were prepared with three fractional viabilities and provided with 17 O 2 . Cellular metabolism of 17 O 2 into nascent mitochondrial water (H 2 17 O) was monitored by 17 O-MRS and, from the measured data, OCR was calculated. For comparison, OCR was simultaneously measured on a separate, but equivalent sample of cells with a well-established stirred microchamber technique. OCR measured by 17 O-MRS agreed well with measurements made in the stirred microchamber device. These studies confirm that 17 O-MRS can quantify TEG viability noninvasively. Biotechnol. Bioeng. 2017;114: 1118-1121. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Techniques to monitor the oxygen partial pressure (pO 2 ) within implanted tissue-engineered grafts (TEGs) are critically necessary for TEG development, but current methods are invasive and inaccurate. In this study, we developed an accurate and noninvasive technique to monitor TEG pO 2 utilizing proton ( 1 H) or fluorine ( 19 F) magnetic resonance spectroscopy (MRS) relaxometry. The value of the spin-lattice relaxation rate constant (R 1 ) of some biocompatible compounds is sensitive to dissolved oxygen (and temperature), while insensitive to other external factors. Through this physical mechanism, MRS can measure the pO 2 of implanted TEGs. We evaluated six potential MRS pO 2 probes and measured their oxygen and temperature sensitivities and their intrinsic R 1 values at 16.4 T. Acellular TEGs were constructed by emulsifying porcine plasma with perfluoro-15-crown-5-ether, injecting the emulsion into a macroencapsulation device, and cross-linking the plasma with a thrombin solution. A multiparametric calibration equation containing R 1 , pO 2 , and temperature was empirically generated from MRS data and validated with fiber optic (FO) probes in vitro. TEGs were then implanted in a dorsal subcutaneous pocket in a murine model and evaluated with MRS up to 29 days postimplantation. R 1 measurements from the TEGs were converted to pO 2 values using the established calibration equation and these in vivo pO 2 measurements were simultaneously validated with FO probes. Additionally, MRS was used to detect increased pO 2 within implanted TEGs that received supplemental oxygen delivery. Finally, based on a comparison of our MRS data with previously reported data, ultra-high-field (16.4 T) is shown to have an advantage for measuring hypoxia with 19 F MRS. Results from this study show MRS relaxometry to be a precise, accurate, and noninvasive technique to monitor TEG pO 2 in vitro and in vivo., Competing Interests: Statement No competing financial interests exist.

Diabetes is a major health problem worldwide, and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges, and young pigs (3-6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (~3 months; 25 kg Yorkshire pigs) immediately following procurement or after 24 h of hypothermic machine perfusion (HMP) preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology, and in vivo function. Islet yields were highest from HMP-preserved pancreata (2,242 ± 449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 μm), and purity was on average 63 ± 6%. Morphologically, islets appeared as clusters on day 0, loosely disaggregated structures at day 1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day 6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki-67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 175 ± 16 nmol/min/mg DNA, and physiological function was intact by glucose stimulation after 6-8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) of transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation require specialized conditions. These immature islets undergo plasticity in culture and form fully functional multicellular structures. Further development of this method for culturing immature porcine islets is expected to generate small pancreatic tissue-derived organoids termed "pancreatites," as a therapeutic product from juvenile pigs for xenotransplantation and diabetes research.

In this demonstration, spheroids formed from the β-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Beta-cell replacement therapy is a promising approach for the treatment of diabetes but is currently limited by the human islet availability and by the need for systemic immunosuppression. Tissue engineering approaches that will enable the utilization of islets or β-cells from alternative sources (such as porcine islets or human stem cell derived beta cells) and minimize or eliminate the need for immunosuppression have the potential to address these critical limitations. However, tissue engineering approaches are critically hindered by the device size (similar to the size of a large flat screen television) required for efficacy in humans. The primary factor dictating the device size is the oxygen availability to islets to support their viability and function (glucose-stimulated insulin secretion [GSIS]). GSIS is affected (inhibited) at a much higher oxygen partial pressure [pO2] than that of viability (e.g. 10 mmHg as opposed to 0.1 mmHg). Enhanced oxygen supply (higher pO2) than what is available in vivo at transplant sites can have a profound effect on the required device size (potentially reduce it to the size of a postage stamp). This paper summarizes key information on the effect of oxygen on islet viability and function within immunoisolation devices and describes the potential impact of enhanced oxygen supply to devices in vivo on device size reduction.

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

Intraportal islet transplantation (IT) is not widely utilized as a treatment for type 1 diabetes. Oxygenation of the intraportally transplanted islet has not been studied extensively. We present a diffusion-reaction model that predicts the presence of an anoxic core and a larger partly functional core within intraportally transplanted islets. Four variables were studied: islet diameter, islet fractional viability, external oxygen partial pressure ( P ) (in surrounding portal blood), and presence or absence of a thrombus on the islet surface. Results indicate that an islet with average size and fractional viability exhibits an anoxic volume fraction (AVF) of 14% and a function loss of 72% at a low external P . Thrombus formation increased AVF to 30% and function loss to 92%, suggesting that the effect of thrombosis may be substantial. External P and islet diameter accounted for the greatest overall impact on AVF and loss of function. At our institutions, large human alloislets (>200 μ m diameter) account for ~20% of total islet number but ~70% of total islet volume; since most of the total transplanted islet volume is accounted for by large islets, most of the intraportal islet cells are likely to be anoxic and not fully functional.

Background: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity., Methods: Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis., Results: Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72)., Conclusions: Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

Porcine islet xenotransplantation is a promising alternative to human islet allotransplantation. Porcine pancreas cooling needs to be optimized to reduce the warm ischemia time (WIT) following donation after cardiac death, which is associated with poorer islet isolation outcomes. This study examines the effect of four different cooling Methods on core porcine pancreas temperature (n = 24) and histopathology (n = 16). All Methods involved surface cooling with crushed ice and chilled irrigation. Method A, which is the standard for porcine pancreas procurement, used only surface cooling. Method B involved an intravascular flush with cold solution through the pancreas arterial system. Method C involved an intraductal infusion with cold solution through the major pancreatic duct, and Method D combined all three cooling Methods. Surface cooling alone (Method A) gradually decreased core pancreas temperature to <10 °C after 30 min. Using an intravascular flush (Method B) improved cooling during the entire duration of procurement, but incorporating an intraductal infusion (Method C) rapidly reduced core temperature 15-20 °C within the first 2 min of cooling. Combining all methods (Method D) was the most effective at rapidly reducing temperature and providing sustained cooling throughout the duration of procurement, although the recorded WIT was not different between Methods (P = 0.36). Histological scores were different between the cooling Methods (P = 0.02) and the worst with Method A. There were differences in histological scores between Methods A and C (P = 0.02) and Methods A and D (P = 0.02), but not between Methods C and D (P = 0.95), which may highlight the importance of early cooling using an intraductal infusion. In conclusion, surface cooling alone cannot rapidly cool large (porcine or human) pancreata. Additional cooling with an intravascular flush and intraductal infusion results in improved core porcine pancreas temperature profiles during procurement and histopathology scores. These data may also have implications on human pancreas procurement as use of an intraductal infusion is not common practice., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Porcine islet xenotransplantation is emerging as a potential alternative for allogeneic clinical islet transplantation. Optimization of porcine islet isolation in terms of yield and quality is critical for the success and cost-effectiveness of this approach. Incomplete pancreas distention and inhomogeneous enzyme distribution have been identified as key factors for limiting viable islet yield per porcine pancreas. The aim of this study was to explore the utility of magnetic resonance imaging (MRI) as a tool to investigate the homogeneity of enzyme delivery in porcine pancreata. Traditional and novel methods for enzyme delivery aimed at optimizing enzyme distribution were examined. Pancreata were procured from Landrace pigs via en bloc viscerectomy. The main pancreatic duct was then cannulated with an 18-g winged catheter and MRI performed at 1.5-T. Images were collected before and after ductal infusion of chilled MRI contrast agent (gadolinium) in physiological saline. Regions of the distal aspect of the splenic lobe and portions of the connecting lobe and bridge exhibited reduced delivery of solution when traditional methods of distention were utilized. Use of alternative methods of delivery (such as selective re-cannulation and distention of identified problem regions) resolved these issues, and MRI was successfully utilized as a guide and assessment tool for improved delivery. Current methods of porcine pancreas distention do not consistently deliver enzyme uniformly or adequately to all regions of the pancreas. Novel methods of enzyme delivery should be investigated and implemented for improved enzyme distribution. MRI serves as a valuable tool to visualize and evaluate the efficacy of current and prospective methods of pancreas distention and enzyme delivery., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Background: The amount and condition of exocrine impurities may affect the quality of islet preparations, especially during culture. In this study, the objective was to determine the oxygen demand and viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture., Method: We compared the oxygen consumption rate (OCR) normalized to DNA (OCR/DNA, a measure of fractional viability in units of nmol/min/mg DNA), and the percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture., Results: The mean (±SE) OCR/DNA was 74.0 ± 11.7 units higher for acinar (vs islet) tissue on the day of isolation (n = 16, P < .0001), but 25.7 ± 9.4 units lower after 1 day (n = 8, P = .03), 56.6 ± 11.5 units lower after 2 days (n = 12, P = .0004), and 65.9 ± 28.7 units lower after 8 days (n = 4, P = .2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n = 6). DNA recovery decreased to 24 ± 7% for acinar and 75 ± 8% for islets (P = .002). Similarly, OCR recovery decreased to 16 ± 3% for acinar and remained virtually constant for islets (P = .005)., Conclusion: Differences in the metabolic profile of acinar and islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-islet transplantation culture protocols., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Background: The shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment)., Method: IE were isolated from two manufacturing centers and shipped in 10-cm(2) surface area SRM vessels in temperature- and pressure-controlled containers to a distant center after at least 2 days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a microcentrifuge tube negative control (NC). LD was designed to mimic the standard culture density for IE preparations (200 IE/cm(2)), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1-3 vessels. Upon receipt, islets were assessed for viability (measured by oxygen consumption rate normalized to DNA content [OCR/DNA)]), quantity (measured by DNA), and, when possible, potency and function (measured by dynamic glucose-stimulated insulin secretion measurements and transplants in immunodeficient B6 Rag(+/-) mice). Postshipment OCR/DNA was not reduced in HD vs LD and was substantially reduced in the NC condition. HD islets exhibited normal function postshipment. Based on the data, we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Stress conditions generated throughout pancreatic islet processing initiate the activation of pro-inflammatory pathways and beta-cell destruction. Our goal is to identify relevant and preferably beta-specific markers to assess the activation of beta-cell stress and apoptotic mechanisms, and therefore the general quality of the islet preparation prior to transplantation. Protein expression and activation were analyzed by Western blotting and kinase assays. ATP measurements were performed by a luminescence-based assay. Oxygen consumption rate (OCR) was measured based on standard protocols using fiber optic sensors. Total RNA was used for gene expression analyzes. Our results indicate that pancreas digestion initiates a potent stress response in the islets by activating two stress kinases, c-Jun N-terminal Kinase (JNK) and p38. JNK1 protein levels remained unchanged between different islet preparations and following culture. In contrast, levels of JNK3 increased after islet culture, but varied markedly, with a subset of preparations bearing low JNK3 expression. The observed changes in JNK3 protein content strongly correlated with OCR measurements as determined by the Spearman's rank correlation coefficient rho [Formula: see text] in the matching islet samples, while inversely correlating with c-fos mRNA expression [Formula: see text]. In conclusion, pancreas digestion recruits JNK and p38 kinases that are known to participate to beta-cell apoptosis. Concomitantly, the islet isolation alters JNK3 and c-fos expression, both strongly correlating with OCR. Thus, a comparative analysis of JNK3 and c-fos expression before and after culture may provide for novel markers to assess islet quality prior to transplantation. JNK3 has the advantage over all other proposed markers to be islet-specific, and thus to provide for a marker independent of non-beta cell contamination.

Background: Human islet allotransplantation for the treatment of type 1 diabetes is in phase III clinical trials in the U.S. and is the standard of care in several other countries. Current islet product release criteria include viability based on cell membrane integrity stains, glucose-stimulated insulin release, and islet equivalent (IE) dose based on counts. However, only a fraction of patients transplanted with islets that meet or exceed these release criteria become insulin independent following 1 transplant. Measurements of islet oxygen consumption rate (OCR) have been reported as highly predictive of transplant outcome in many models., Method: In this article we report on the assessment of clinical islet allograft preparations using OCR dose (or viable IE dose) and current product release assays in a series of 13 first transplant recipients. The predictive capability of each assay was examined and successful graft function was defined as 100% insulin independence within 45 days post-transplant., Results: OCR dose was most predictive of CTO. IE dose was also highly predictive, while glucoses stimulated insulin release and membrane integrity stains were not., Conclusion: OCR dose can predict CTO with high specificity and sensitivity and is a useful tool for evaluating islet preparations prior to clinical human islet allotransplantation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Replacement of β-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability., Methods: After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity)., Results: Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media., Conclusions: Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The islet size distribution in a preparation may contribute to islet transplant outcomes. At the same islet equivalent (IE) dose, larger islets may exhibit poorer therapeutic value and this may be because of oxygen diffusion limitations that worsen in proportion to islet size., Methods: To test this hypothesis, we studied the impact of islet size index (ISI) and other islet product characteristics on outcomes after islet autotransplant (IAT) in recipients receiving a marginal islet dose (2000-4999 IEs per kg body weight) from January 1, 2009 to June 11, 2012, at the University of Minnesota (n=58). ISI was defined as the number of IE divided by the number of islet particles (IPs) in a preparation; an ISI less than 1 indicates a mean islet diameter that is less than 150 μm. The primary post-IAT outcome was 6-month insulin use status., Results: Logistic regression analysis indicate that IPs/kg (P=0.001), IEs/kg (P=0.019), total IPs transplanted (P=0.040), and ISI (P=0.074) were most strongly correlated with the primary outcome. The ISI (mean±standard error) was lower for recipients achieving insulin independence at 6 months (0.71±0.05) versus those partially (0.83±0.05) or completely (1.00±0.07) insulin dependent. The combination of islet dose (expressed as units IPs/kg) and ISI exhibited a sensitivity of 75% and specificity of 74% in predicting insulin independence in this population of patients., Conclusion: Islet autotransplant recipients of a marginal islet doses were more likely to achieve insulin independence when transplanted with a greater number of smaller islets.

Islet transplantation (IT) is a promising therapy for the treatment of diabetes. The large number of islets required to achieve insulin independence limit its cost-effectiveness and the number of patients who can be treated. It is believed that >50% of islets are lost in the immediate post-IT period. Poor oxygenation in the early post-IT period is recognized as a possible reason for islet loss and dysfunction but has not been extensively studied. Several key variables affect oxygenation in this setting, including (1) local oxygen partial pressure (pO(2)), (2) islet oxygen consumption, (3) islet size (diameter, D), and (4) presence or absence of thrombosis on the islet surface. We discuss implications of oxygen-limiting conditions on intraportal islet viability and function. Of the 4 key variables, the islet size appears to be the most important determinant of the anoxic and nonfunctional islet volume fractions. Similarly, the effect of thrombus formation on the islet surface may be substantial. At the University of Minnesota, average size distribution data from clinical alloislet preparations (n = 10) indicate that >150-µm D islets account for only ~30% of the total islet number, but >85% of the total islet volume. This suggests that improved oxygen supply to the islets may have a profound impact on islet survivability and function since most of the β-cell volume is within large islets which are most susceptible to oxygen-limiting conditions. The assumption that the liver is a suitable islet transplant site from the standpoint of oxygenation should be reconsidered., (© 2014 Diabetes Technology Society.)

Islet transplantation is a promising treatment for type 1 diabetes, but despite the successes, existing challenges prevent widespread application. Ischemia, occurring during pancreas preservation and isolation, as well as after islet transplantation, decreases islet viability and function. We hypothesized that the liposomal delivery of adenosine triphosphate (ATP) could prevent the loss of cell viability during an ischemic insult. In this work we use a model β cell line, INS-1 to probe the liposome/cell interactions and examined the ability of liposomes functionalized with the fibronectin-mimetic peptide PR&#95;b to facilitate the delivery of ATP to ischemic β cells. We demonstrate that PR&#95;b increases the binding and internalization of liposomes to the β cells. Unexpectedly, when comparing the ability of PR&#95;b liposomes with and without ATP to protect INS-1 cells from ischemia we found that both formulations increased cell survival. By probing the functional activity of ischemic cells treated with PR&#95;b functionalized liposomes with and without ATP we find that both lipids and ATP play a role in maintaining cell metabolic activity after an ischemic insult and preventing cell necrosis. This approach may be beneficial for preventing ischemia related damage to islet cells, especially in the organ preservation stage.

Background: The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 °C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs., Methods: Porcine islets were isolated from three donors and cultured at 37 °C for 1 day, and then under three different conditions: 37 °C for 6 days (condition A); 22 °C for 6 days (condition B); or 22 °C for 5 days followed by 37 °C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers., Results: Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159 nmol/min·mgDNA before culture, and 259-291, 204-212, and 207-228 nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 h at 37 °C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22 °C to those in islets cultured at 37 °C. Results were consistent among the preparations from the three donors., Conclusions: Culture of porcine islets at 37 °C provides benefits over culture at 22 °C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress., (© 2013 John Wiley & Sons A/S.)

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications.