W. E. Scott

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.

Pig Pancreas Anatomy: Implications for Pancreas Procurement, Preservation, and Islet Isolation

Background. Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. The limited human islet supply from cadavers and poor islet yield and quality remain substantial impediments to progress in the field. Use of porcine islets holds great promise for large-scale application of islet transplantation. Consistent isolation of porcine islets is dependent on advances in pancreas procurement, pancreas preservation, and islet isolation, requiring detailed knowledge of the porcine pancreatic anatomy. The primary aim of this study was to describe the vascular and ductal anatomy of the porcine pancreas to guide and improve organ preservation and enzyme perfusion. Methods. Pancreata were removed by en bloc viscerectomy from 65 female Landrace pigs. Results. Fifteen percentage of organs exhibited inconsistent vascular branching from the celiac trunk. All organs showed uniform patterns of branching at the superior mesenteric artery. The superior and inferior mesenteric veins merged to become the portal vein in all but one case in which the inferior mesenteric vein drained into the splenic vein. Ninety-seven percent of pancreata had three lobes: duodenal lobe (DL), connecting lobe (CL), and splenic lobe (SL); 39% demonstrated ductal communication between the CL and the other two lobes; 50% had ductal communication only between the CL and duodenal lobe; and 11% presented other types of ductal delineation. Conclusions. Accounting for the variations in vascular and ductal anatomy, as detailed in this study, will facilitate development of protocols for preservation, optimal enzyme administration, and pancreas distention and digestion, and will ultimately lead to substantial improvements in isolation outcomes.

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.

Persufflation (or Gaseous Oxygen Perfusion) as a Method of Organ Preservation

Improved preservation techniques have the potential to improve transplant outcomes by better maintaining donor organ quality and by making more organs available for allotransplantation. Persufflation, (PSF, gaseous oxygen perfusion) is potentially one such technique that has been studied for over a century in a variety of tissues, but has yet to gain wide acceptance for a number of reasons. A principal barrier is the perception that ex vivo PSF will cause in vivo embolization post-transplant. This review summarizes the extensive published work on heart, liver, kidney, small intestine and pancreas PSF, discusses the differences between anterograde and retrograde PSF, and between PSF and other conventional methods of organ preservation (static cold storage, hypothermic machine perfusion). Prospective implications of PSF within the broader field of organ transplantation, and in the specific application with pancreatic islet isolation and transplant are also discussed. Finally, key issues that need to be addressed before PSF becomes a more widely utilized preservation strategy are summarized and discussed.

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.

Supplements in human islet culture: human serum albumin is inferior to fetal bovine serum.

Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HSA as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

Quality of Isolated Pig Islets Is Improved Using Perfluorohexyloctane for Pancreas Storage in a Split Lobe Model

Pancreas transportation between donor center and islet production facility is frequently associated with prolonged ischemia impairing islet isolation and transplantation outcomes. It is foreseeable that shipment of pig pancreases from distant centralized biosecure breeding facilities to institutes that have a long-term experience in porcine islet isolation is essentially required in future clinical islet xenotransplantation. Previously, we demonstrated that perfluorohexyloctan (F6H8) is significantly more efficient to protect rat and human pancreata from ischemically induced damage compared to perfluorodecalin (PFD). To evaluate the effect of F6H8 on long-term stored pig pancreases in a prospective study, we utilized the split lobe model to minimize donor variability. Retrieved pancreases were dissected into the connecting and splenic lobe, intraductally flushed with UW solution and immersed alternately in either preoxygenated F6H8 or PFD for 8–10 h. Prior to pancreas digestion, the intrapancreatic pO2 and the ratio of ATP-to-inorganic phosphate was compared utilizing 31P-NMR spectroscopy. Isolated islets were cultured for 2–3 days at 37°C and subjected to quality assessment. Pancreatic lobes stored in preoxygenated F6H8 had a significantly higher intrapancreatic pO2 compared to pancreata in oxygen-precharged PFD (10.11 ± 3.87 vs. 1.64 ± 1.13 mmHg, p < 0.05). This correlated with a higher ATP-to-inorganic phosphate ratio (0.30 ± 0.04 vs. 0.14 ± 0.01). No effect was observed concerning yield and purity of freshly isolated islets. Nevertheless, a significantly improved glucose-stimulated insulin response, increased viability and postculture survival (57.2 ± 5.7 vs. 39.3 ± 6.4%, p < 0.01) was measured in islets isolated from F6H8-preserved pancreata. The present data suggest that F6H8 does not increase islet yield but improves quality of pig islets isolated after prolonged cold ischemia.