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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.)

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.

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 considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets., (© 2013 John Wiley & Sons A/S.)

Background: Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of α-1 antitrypsin (A1AT)., Methods: Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations. The effect of proteases on ultrastructure of islets and β-cell insulin granules were examined by transmission electron microscopy. The number of insulin granules and insulin-labeled immunogold particles were counted. The in vivo effect of proteases on islet function was studied by transplanting acinar cells adjacent to islet grafts in diabetic mice. The effects of A1AT culture supplementation on protease activity, insulin levels, and islet function were assessed in pure and impure islets., Results: Islet loss after culture was significantly higher in impure relative to pure preparations (30% vs. 14%, P<0.04). Lower islet purity was associated with increased protease activity and decreased insulin levels in culture supernatants. Reduced β-cell insulin granules and insulin degradation by proteases were confirmed by transmission electron microscopy. Transplantations in mice showed delayed islet graft function when acinar cells were transplanted adjacent to the islets under the kidney capsule. Supplementation of A1AT to impure islet cultures maintained islet cell mass, restored insulin levels, and preserved islet functional integrity., Conclusion: Culture of impure human islet fractions in the presence of A1AT prevents insulin degradation and improves islet recovery.

Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with (31)P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by (13)C NMR isotopomer analysis of the fate of [U-(13)C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We sought to determine the long-term outcomes in type 1 diabetic recipients of intraportal alloislet transplants on a modified immunosuppressive protocol. Six recipients with hypoglycemia unawareness received one to two islet infusions. Induction therapy was with antithymocyte globulin (ATG) plus etanercept for tumor necrosis factor-alpha blockade. Recipients received cyclosporine and everolimus for maintenance immunosuppression for the first year posttransplant, with mycophenolic acid or mycophenolate mofetil subsequently substituted for everolimus. Recipients have been followed for 1173 +/- 270 days since their last infusion for islet graft function (insulin independence, hemoglobin A(1c) levels and C-peptide production) and for adverse events associated with the study protocol. Of the six recipients, five were insulin-independent at 1 year, and four continue to be insulin-independent at a mean of 3.4 +/- 0.4 years posttransplant. None of the six recipients experienced recurrence of severe hypoglycemia. Measured glomerular filtration rate decreased from 110.5 +/- 21.2 mL/min/1.73 m(2) pretransplant to 82.6 +/-19.1 mL/min/1.73 m(2) at 1 year posttransplant. In conclusion, islet transplants restored insulin independence for a mean of >3 years in four of six recipients treated with ATG and etanercept induction therapy and with cyclosporine and, initially, everolimus for maintenance. Our results suggest this immunosuppressive protocol may allow long-term graft survival.

Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process.

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion., (Copyright 1999 John Wiley & Sons, Inc.)

We investigated the effects of glucose on the rates of oxygen consumption (OCR) and insulin secretion (ISR) by betaHC9 cells derived from mouse pancreatic islets with beta-cell hyperplasia. Our results demonstrate that the OCR by betaHC9 cells incubated in nutrient-rich DMEM is unaffected by glucose (0-25 mM), is dissociated from the ISR (which increases with the addition of glucose), and is always higher than that measured in PBS. Glucose (25 mM) increases both the OCR and ISR when added to nutrient-free PBS. On the basis of results presented here, we suggest that, contrary to the current consensus, the observed increases in the OCR by beta-cells upon addition of glucose to nutrient-free buffers may be unrelated to the process of glucose-stimulated insulin secretion (GSIS) and, instead, related to nutrient starvation. We believe that a reevaluation of the implication of changes in OCR upon glucose stimulation in the process of GSIS is warranted and that OCR and ISR measurements should be performed in more physiological media to avoid nutrient starvation artifacts.

Understanding the biochemical events associated with glucose-stimulated insulin secretion by pancreatic beta cells is of importance in gaining insight into both the pathophysiology of diabetes and the development of tissue-engineered bioartificial pancreatic substitutes. We have investigated the effects of glucose concentration on the bioenergetic status and on the metabolic and secretory functions exhibited by mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cells entrapped in APA beads constitute a possible implantable bioartificial pancreas for the long-term treatment of insulin-dependent diabetes mellitus. Our results show that, in entrapped betaTC3 cells, the oxygen consumption rate and the intracellular nucleotide triphosphate levels are unaffected by a step change in glucose concentration from 16 mM to 0 mM for 4.5 h and then back to 16 mM. The intracellular Pi level and the ammonia production rate were doubled, while insulin secretion was decreased 10-fold, upon switching from 16 mM to 0 mM glucose. The implications of these findings in the context of pancreatic beta cell biochemistry and the mechanism of the 'Fuel Hypothesis' are discussed.

A promising method for diabetes treatment is the implantation of immunoisolated cells secreting insulin in response to glucose. Cell availability limits the application of this approach at a medically-relevant scale. We explore the use of transformed cells that can be grown to large homogeneous populations in developing artificial pancreatic tissues. We also investigate the use of NMR in evaluating, non-invasively, cellular bioenergetics in the tissue environment. The system employed in this study consisted of mouse insulinoma beta TC3 cells entrapped in calcium alginate/poly-L-lysine (PPL)/alginate beads. The PPL layer imposed a molecular weight cutoff of approximately 60 kDa, allowing nutrients and insulin to diffuse through but excluding high molecular weight antibodies and cytotoxic cells of the host. We fabricated a radiofrequency coil that can be double-tuned to 1H and 31P, and an NMR-compatible perfusion bioreactor and support circuit that can maintain cells viable during prolonged studies. The bioreactor operated differentially, was macroscopically homogeneous and allowed the acquisitions of 1H images and 31P NMR spectra in reasonable time intervals. Results indicated that entrapment had little effect on cell viability; that insulin secretion from beads was responsive to glucose; and that the bioenergetics of perfused, entrapped cells were not grossly different from those of cells never subjected to the immobilization procedure. These findings offer promise for developing an artificial pancreatic tissue for diabetes treatment based on continuous cell lines.

Animal cells from endocrine glands have potential applications in bioprocessing, for the production of hormones, enzymes, possibly also recombinant proteins, and in tissue engineering, for the development of immunoisolated, implantable devices for long-term treatment of endocrine disorders. Immunoisolation can be achieved by surrounding cells with a biocompatible polymer which allows diffusion of nutrients and metabolites, including hormones, but excludes higher molecular weight antibodies and cytotoxic cells. Primary hormone-secreting cells cannot be effectively amplified in culture, so the large-scale application of implantable systems based on such cells is limited by cell availability. In this study, we conducted an initial assessment of the feasibility of using transformed, continuous cell lines in immunoisolated devices. The model system employed consisted of mouse pituitary tumor AtT-20 cells which secrete recombinant proinsulin and an insulin-like peptide and exhibit a high growth potential. Cells were cultivated as spheroids in spinner flasks and entrapped as such in alginate/polylysine/alginate beads. Free and entrapped spheroids were propagated in fed-batch, suspension cultures. Entrapment did not significantly affect spheroid metabolism or basal secretion. Entrapped spheroids did not increase in size or number and maintained roughly constant metabolic and basal secretory activities over a 15-day period. Free spheroids in suspension increased in size during the same period, but also maintained constant metabolism and basal secretion, apparently because of a concomitant increase in hypoxic and/or necrotic cells. The potential of using continuous cell lines in the development of bioartificial endocrine organs is discussed.

BackgroundThe 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).MethodIE 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.