Efstathios S. Avgoustiniatos

Human Islet Oxygen Consumption Rate and DNA Measurements Predict Diabetes Reversal in Nude Mice

There is a need for simple, quantitative and prospective assays for islet quality assessment that are predictive of islet transplantation outcome. The current state‐of‐the‐art athymic nude mouse bioassay is costly, technically challenging and retrospective. In this study, we report on the ability of 2 parameters characterizing human islet quality: (1) oxygen consumption rate (OCR), a measure of viable volume; and (2) OCR/DNA, a measure of fractional viability, to predict diabetes reversal in nude mice. Results demonstrate that the probability for diabetes reversal increases as the grafts OCR/DNA and total OCR increase. For a given transplanted OCR dose, diabetes reversal is strongly dependent on OCR/DNA. The OCR and OCR/DNA (the ‘OCR test’) data exhibit 89% sensitivity and 77% specificity in predicting diabetes reversal in nude mice (n = 86). We conclude that the prospective OCR test can effectively replace the retrospective athymic nude mouse bioassay in assessing human islet quality prior to islet transplantation.

In Situ Electrochemical Oxygen Generation with an Immunoisolation Device

The viability and function of transplanted tissue encapsulated in immunobarrier devices is subject to oxygen transport limitation. In this study, we have designed and used an in situ electrochemical oxygen generator which decomposes water electrolyticaly to provide oxygen to the adjacent planer immunobarrier diffusion chamber. The rate of oxygen generation, which increases linearly with electrical current, was accurately controlled. A theoretical model of oxygen diffusion was also developed and was used to calculate the oxygen profiles in some of the experimental systems. In vitro culture experiments were carried out with βTC3 cells encapsulated in titanium ring devices. The growth and viability of cells with or without in situ oxygen generation was studied. We found that under otherwise similar culturing conditions, the thickness of the cell layer and the viability of cells was the highest in devices cultured in stirred media with oxygen generation, even though the thickness had not reached the theoretically predicted value, and lowest in those unstirred and without oxygen generation.

OXYGEN PERSUFFLATION INCREASES PANCREATIC ATP LEVELS AND VIABLE ISLET YIELD FOLLOWING 24 HOURS PRESERVATION COMPARED WITH THE TWO-LAYER METHOD (TLM): 1196

W.E. Scott III1, E.S. Avgoustiniatos2, J. Ferrer-Fabrega2, B.P. Weegman2, V.A. Kircner2, T. Anazawa3, M.D. Rizzari4, L.S. Kidder2, S.A. Stein2, S. Matsumoto2, J.J. Stone2, T.M. Suszynski5, T.C. Aasheim2, B.E. Hammer6, A.N. Balamurugan2, T.D. O’Brien7, M.P. Murtaugh8, L.A. Tempelman9, D.E. Sutherland2, B.J. Hering2, K.K. Papas2 1, , St. Paul/MN/UNITED STATES OF AMERICA, 2Surgery, University of Minnesota, Minneapolis/UNITED STATES OF AMERICA, 3Organ Regenerative Surgery, Fukushima Medical University, Fukushima/ Fukushima/JAPAN, 4Department Of Surgery, University of Minnesota, Minneapolis/Minnesota/UNITED STATES OF AMERICA, 5Department Of Surgery, University of Minnesota, Minneapolis/MN/UNITED STATES OF AMERICA, 6Radiology, University of Minnestoa, Minneapolis/MN/UNITED STATES OF AMERICA, 7Veterinary Population Medicine, University of Minnestoa, St. Paul/MN/UNITED STATES OF AMERICA, 8Veterinary Biosciences, University of Minnestoa, St. Paul/MN/UNITED STATES OF AMERICA, 9, Giner Inc., Newton/MA/UNITED STATES OF AMERICA

Pancreas Oxygen Persufflation Increases ATP Levels as Shown by Nuclear Magnetic Resonance

BACKGROUND Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion. METHODS Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for (31)P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:P(i)) and for assessing PSF homogeneity by MRI. RESULTS Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:P(i) levels in rat pancreata but not in larger porcine pancreata. ATP:P(i) levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:P(i) was elevated to levels similar to those observed in rat pancreata. CONCLUSION The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool.

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.

Persufflation Improves Pancreas Preservation When Compared With the Two-Layer Method

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.

Real-Time Noninvasive Assessment of Pancreatic ATP Levels During Cold Preservation

31P-NMR spectroscopy was utilized to investigate rat and porcine pancreatic ATP:P(i) ratios to assess the efficacy of existing protocols for cold preservation (CP) in maintaining organ quality. Following sacrifice, rat pancreata were immediately excised or left enclosed in the body for 15 minutes of warm ischemia (WI). After excision, rat pancreata were stored at 6 degrees C to 8 degrees C using histidine-tryptophan-ketoglutarate solution (HTK) presaturated with air (S1), HTK presaturated with O2 (S2), or the HTK/perfluorodecalin two-layer method (TLM) with both liquids presaturated with O2 (S3). 31P-NMR spectra were sequentially collected at 3, 6, 9, 12, and 24 hours of CP from pancreata stored with each of the three protocols examined. The ATP:Pi ratio for rat pancreata exposed to 15 minutes of WI and stored with S3 increased during the first 9 hours of CP, approaching values observed for organs procured with no WI. A marked reduction in the ATP:Pi ratio was observed beyond 12 hours of CP with S3. After 6 hours of CP, the ATP:Pi ratio was highest for S3, substantially decreased for S2, and below detection for S1. In sharp contrast to the rat model, ATP was barely detectable in porcine pancreata exposed to minimal warm ischemia (<15 minutes) stored with the TLM regardless of CP time. We conclude that 31P-NMR spectroscopy is a powerful tool that can be used to (1) noninvasively evaluate pancreata prior to islet isolation, (2) assess the efficacy of different preservation protocols, (3) precisely define the timing of reversible versus irreversible damage, and (4) assess whether intervention will extend this timing.

Continuous Real-time Viability Assessment of Kidneys Based on Oxygen Consumption

BACKGROUND Current ex vivo quality assessment of donor kidneys is limited to vascular resistance measurements and histological analysis. New techniques for the assessment of organ quality before transplantation may further improve clinical outcomes while expanding the depleted deceased-donor pool. We propose the measurement of whole organ oxygen consumption rate (WOOCR) as a method to assess the quality of kidneys in real time before transplantation. METHODS Five porcine kidneys were procured using a donation after cardiac death (DCD) model. The renal artery and renal vein were cannulated and the kidney connected to a custom-made hypothermic machine perfusion (HMP) system equipped with an inline oxygenator and fiber-optic oxygen sensors. Kidneys were perfused at 8 degrees C, and the perfusion parameters and partial oxygen pressures (pO(2)) were measured to calculate WOOCR. RESULTS Without an inline oxygenator, the pO(2) of the perfusion solution at the arterial inlet and venous outlet diminished to near 0 within minutes. However, once adequate oxygenation was provided, a significant pO(2) difference was observed and used to calculate the WOOCR. The WOOCR was consistently measured from presumably healthy kidneys, and results suggest that it can be used to differentiate between healthy and purposely damaged organs. CONCLUSIONS Custom-made HMP systems equipped with an oxygenator and inline oxygen sensors can be applied for WOOCR measurements. We suggest that WOOCR is a promising approach for the real-time quality assessment of kidneys and other organs during preservation before transplantation.

Devices and Methods for Maintenance of Temperature and Pressure During Islet Shipment

UNLABELLED Exposure to extreme temperatures and pressure fluctuations during shipment by air may have a detrimental impact on islet quality. In this study, we sought to assess the ability of methods and devices to provide better control of the internal environment of islet shipping containers in terms of temperature and pressure. METHODS Experimental islet shipping containers were packed with 21 panels of commercially available TCP Phase 22 Phase Change Material (TCP). The containers were then exposed for at least 15 hours to three constant external temperature conditions, namely, -20 degrees C, 4 degrees C, and 40 degrees C, and then evaluated for their ability to maintain an internal temperature close to the desired value of 22 degrees C. Custom-designed pressure regulated gyroscopic shipping containers (PRGSC) placed in a vacuum chamber were exposed to an absolute pressure of 250 mm Hg (substantially lower than that experienced during shipment by air) for 25 minutes to assess their ability to control internal pressure under vacuum. Electronic data loggers were used to monitor internal and external temperatures and pressures under all conditions. RESULTS Twenty-one TCP panels placed in a single islet shipping container were able to maintain the internal temperature between 17 degrees C and 24 degrees C for a minimum of 15 hours at all three external temperatures. The PRGSC tested were able to maintain a constant internal pressure of 760 mm Hg when exposed to vacuum. CONCLUSIONS Our results demonstrated that the use of containers equipped with TCP and PRGSC exert excellent environmental control over islet shipments by minimizing temperature and eliminating pressure fluctuations.

Commercially Available Gas-Permeable Cell Culture Bags May Not Prevent Anoxia in Cultured or Shipped Islets

Prolonged anoxia has deleterious effects on islets. Gas-permeable cell culture devices can be used to minimize anoxia during islet culture and especially during shipment when elimination of gas-liquid interfaces is required to prevent the formation of damaging gas bubbles. Gas-permeable bags may have several drawbacks, such as propensity for puncture and contamination, difficult islet retrieval, and significantly lower oxygen permeability than silicone rubber membranes (SRM). We hypothesized that oxygen permeability of bags may be insufficient for islet oxygenation. We measured oxygen transmission rates through the membrane walls of three different types of commercially available bags and through SRM currently used for islet shipment. We found that the bag membranes have oxygen transmission rates per unit area about 100-fold lower than SRM. We solved the oxygen diffusion-reaction equation for 150-microm diameter islets seeded at 3000 islet equivalents per cm2, a density adequate to culture and ship an entire human or porcine islet preparation in a single gas-permeable device, predicting that about 40% of the islet volume would be anoxic at 22 degrees C and about 70% would be anoxic at 37 degrees C. Islets of larger size or islets accumulated during shipment would be even more anoxic. The model predicted no anoxia in islets similarly seeded in devices with SRM bottoms. We concluded that commercially available bags may not prevent anoxia during islet culture or shipment; devices with SRM bottoms are more suitable alternatives.