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

Oxygen diffusion limitations in pancreatic islet culture and immunoisolation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2002.Includes bibliographical references.Data for oxygen consumption by rat islets of Langerhans in a batch microreactor were fitted using a numerical solution of the transient oxygen diffusion-reaction equation. Average best-fit values were 3.1 +/- 0.7 x 10-8 mol/cm3-s for the maximum oxygen consumption rate Vmax in aminoacid-free media and 1.2 +/- 0.4 x 10-14 mol/cm-mm Hg-s for the oxygen permeability in islet tissue. These parameter values, along with a 30-60% positive correction for the presence of aminoacids in Vmax, were used to predict oxygen profiles inside and around islets under perifusion, culture, and immunoisolation conditions. The difference between Michaelis-Menten and zero-order kinetics and the role of the necrosis process in modeling of oxygen profiles were found to increase with the severity of hypoxia. Internal and external diffusion limitations were characterized for homogeneously dispersed tissue. Oxygen profiles were determined with finite differences in perifused rat islets for which second-phase insulin secretion data were available. Data were fitted with ad hoc kinetic models describing the effect of local pO2 on insulin secretion. A two-step, one-parameter model that assumed that local insulin secretion rate as a function of local pO2 is first-order for pO2 P* resulted in the best data fit for P* values between 2 and 10 mm Hg, depending on the value of Vmax used. Oxygen profiles were estimated with finite elements for the axi-symmetric problem of single islet culture and the model did an excellent job in predicting loss of viability data, obtained using Trypan blue staining,(cont.) as a function of islet diameter both under normoxic (ambient pO2 = 142 mm Hg) and hypoxic (40 mm Hg) conditions. The model was extended to massive islet culture and the effects of islet surface density and medium depth on viability were characterized and suggestions were made for the improvement of porcine islet culture conditions. In bioartificial pancreas devices we found that there is an optimal islet surface density (NS)opt for which insulin secretion rate is maximized, while secretory efficiency decreases monotonically with tissue density above a critical value. The design tissue density must be chosen in the range between this critical value and (Ns)opt' and its value depends on whether minimization of the device size or the amount of loaded tissue is more important.by Efstathios Spyridon Avgoustiniatos.Ph.D

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

Silicone rubber membrane devices permit islet culture at high density without adverse effects

IntroductionConventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200 IE/cm2. To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen.MethodsOxygenation 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/cm2, as measured by DNA) in both GP and non-GP devices.ResultsIn 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.DiscussionCulturing 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

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

Previously published data showing overlap and correlation of islet equivalent dose with clinical transplant outcome.

<p>Data from Anazawa <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134428#pone.0134428.ref010" target="_blank">10</a>] illustrating that the <i>islet equivalent</i> (<i>IE</i>) <i>dose</i> correlates with the clinical outcome [insulin independence vs. dependence] at 6–12 months following islet autotransplant (IAT). However, the gray region indicates a wide range of <i>IE dose</i> (IE/kg of recipient) that is associated with an uncertain IAT outcome. The second graph shows the receiver operating characteristic (ROC) curve for this previously published data set with the area-under-the-curve (AUC) above.</p

Comparison of demographic, isolation, and quality assessment variables between insulin dependent and independent clinical transplant outcome 6–12 months post-transplant.

<p>Data are means ± standard error or percentage.</p><p>^asignificant difference (p< 0.05) between groups as calculated by a Mann Whitney non-parametric test.</p><p>Comparison of demographic, isolation, and quality assessment variables between insulin dependent and independent clinical transplant outcome 6–12 months post-transplant.</p