16 research outputs found
Islet Assessment for Transplantation
Author Manuscript: 2010 December 1.Purpose of review:
There is a critical need for meaningful viability and potency assays that characterize islet preparations for release prior to clinical islet cell transplantation (ICT). Development, testing, and validation of such assays have been the subject of intense investigation for the past decade. These efforts are reviewed, highlighting the most recent results while focusing on the most promising assays.
Recent Findings:
Assays based on membrane integrity do not reflect true viability when applied to either intact islets or dispersed islet cells. Assays requiring disaggregation of intact islets into individual cells for assessment introduce additional problems of cell damage and loss. Assays evaluating mitochondrial function, specifically mitochondrial membrane potential, bioenergetic status, and cellular oxygen consumption rate (OCR), especially when conducted with intact islets, appear most promising in evaluating their quality prior to ICT. Prospective, quantitative assays based on measurements of OCR with intact islets have been developed, validated and their results correlated with transplant outcomes in the diabetic nude mouse bioassay.
Conclusion:
More sensitive and reliable islet viability and potency tests have been recently developed and tested. Those evaluating mitochondrial function are most promising, correlate with transplant outcomes in mice, and are currently being evaluated in the clinical setting.National Center for Research Resources (U.S.) (Grant)National Institutes of Health (U.S.) (Grant U42 RR 016598–01)National Institutes of Health (U.S.) (Grant RO1-DK063108–01A1)Iacocca FoundationSchott FoundationCarol Olson Memorial Diabetes Research Fun
Oxygenation of the Intraportally Transplanted Pancreatic Islet
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 μm 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
Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation
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
Oxygenation of the Intraportally Transplanted Pancreatic Islet
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 μm 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
Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability
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.Schulze Foundation; Schulze Diabetes Institute, NIH [R44DK072647-03, R01DK068717]; Carol Olson Memorial Diabetes Research Fund; Iacocca Family Foundation; Schott Family Foundation; Kettering Family FoundationThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]
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Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling
BackgroundAll 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.MethodsHuman 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.ResultsPersufflated 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.ConclusionsThese 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
Summary of cases where <i>OCR dose</i> correctly predicted clinical transplant outcome, whereas <i>IE dose</i> did not.
<p>Summary of cases where <i>OCR dose</i> correctly predicted clinical transplant outcome, whereas <i>IE dose</i> did not.</p
Overlap and correlation of islet characterization methods with clinical transplant outcome.
<p>Data from our study illustrating that membrane integrity staining (based on FDA/PI), oxygen consumption rate (OCR) normalized to DNA content (OCR/DNA), and OCR/DNA normalized to the islet size index (ISI) (OCR/DNA/ISI) are not correlated with the clinical outcome [insulin independence vs. dependence] at 6–12 months following islet autotransplant (IAT). However, both <i>islet equivalent</i> (IE) <i>dose</i> and the <i>OCR dose</i> were correlated with post-IAT outcome. The gray region indicates the range of <i>IE</i> and <i>OCR doses</i> that is associated with uncertain IAT outcome. Note that the width of the gray region is much narrower with the <i>OCR dose</i>. The black dotted line represents the calculated cut-off point for clinical outcome (<i>IE dose</i>: 5,794 and <i>OCR dose</i>: 6.23). The second column of graphs represents receiver operating characteristic (ROC) curves for each of the five islet product characteristics from this data set. The area-under-the-curve (AUC) has been calculated for each islet product characteristic and these values are shown above each ROC curve.</p