Porcine Pancreata Research Articles

Ex Vivo Perfusion of Porcine Pancreas and Liver Sourced from Commercial Abattoirs after Circulatory Death as a Research Resource: A Methodological Study.

Machine perfusion (MP) is increasingly used for human transplant organ preservation. The use of MP for research purposes is another opportunity for this technology. The porcine pancreas and liver are similar in anatomical size and function to their human counterparts, making them an excellent resource for research, but they have some important differences from human organs which can influence their research use. In this paper, we describe a technique developed and tested for the retrieval of porcine organs for use in research on perfused viable organs. Whole-organ porcine pancreata and livers were harvested at a commercial abattoir, following standard slaughterhouse processes. The standard slaughterhouse process involved a thoracotomy and mid-line laparotomy, and all the thoracoabdominal organs were removed. The pancreas, fixed in the retroperitoneum, was carefully dissected from its attachments to the surrounding structures, and tissue planes between the pancreas, spleen, duodenum, and colon were meticulously identified and dissected. Vessel exposure and division: The aorta, portal vein (PV), hepatic vein (HV), and hepatic artery (HA) were dissected and isolated, preserving the input and output channels for the liver and pancreas. A distal 3 cm of the aorta was preserved and divided and served as the input for the pancreas perfusions. The liver, PV, HV, and HA were preserved and divided to preserve the physiological channels of the input (PV and HA) and output (HV) for the liver perfusions. The porcine hepatic and pancreas anatomy shares significant resemblance with the gross anatomy found in humans, and this was taken into consideration when designing the perfusion circuitry. The porcine pancreas and spleen shared a common blood supply, with branches arising from the splenic artery. The organs were flushed with cold, heparinised normal saline and transported in a temperature-regulated receptacle maintained at a core temperature between 4 and 8 °C, in line with the standards of static cold storage (SCS), to a dedicated perfusion lab and perfused using our novel perfusion machine with autologous, heparinised porcine blood, also collected at the abattoir.

AP39, a Mitochondrial-Targeted H2S Donor, Improves Porcine Islet Survival in Culture.

The rapid deterioration of transplanted islets in culture is a well-established phenomenon. We recently reported that pancreas preservation with AP39 reduces reactive oxygen species (ROS) production and improves islet graft function. In this study, we investigated whether the addition of AP39 to the culture medium could reduce isolated islet deterioration and improve islet function. Isolated islets from porcine pancreata were cultured with 400 nM AP39 or without AP39 at 37 °C. After culturing for 6–72 h, the islet equivalents of porcine islets in the AP39(+) group were significantly higher than those in the AP39(−) group. The islets in the AP39(+) group exhibited significantly decreased levels of ROS production compared to the islets in the AP39(−) group. The islets in the AP39(+) group exhibited significantly increased mitochondrial membrane potential compared to the islets in the AP39(−) group. A marginal number (1500 IEs) of cultured islets from each group was then transplanted into streptozotocin-induced diabetic mice. Culturing isolated islets with AP39 improved islet transplantation outcomes in streptozotocin-induced diabetic mice. The addition of AP39 in culture medium reduces islet deterioration and furthers the advancements in β-cell replacement therapy.

Pancreas Preservation with a Neutrophil Elastase Inhibitor, Alvelestat, Contributes to Improvement of Porcine Islet Isolation and Transplantation.

For pancreatic islet transplantation, pancreas procurement, preservation, and islet isolation destroy cellular and non-cellular components and activate components such as resident neutrophils, which play an important role in the impairment of islet survival. It has been reported that inhibitors of neutrophil elastase (NE), such as sivelestat and α1-antitrypsin, could contribute to improvement of islet isolation and transplantation. In this study, we investigated whether pancreatic preservation with alvelestat, a novel NE inhibitor, improves porcine islet yield and function. Porcine pancreata were preserved with or without 5 μM alvelestat for 18 h, and islet isolation was performed. The islet yields before and after purification were significantly higher in the alvelestat (+) group than in the alvelestat (−) group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 55% and 5% of diabetic mice in the alvelestat (+) and alvelestat (−) groups, respectively. These results suggest that pancreas preservation with alvelestat improves islet yield and graft function and could thus serve as a novel clinical strategy for improving the outcome of islet transplantation.

Normothermic ex situ pancreas perfusion for the preservation of porcine pancreas grafts

Pancreas transplantation improves and extends the life of patients with insulin-dependent diabetes. Pancreata from extended criteria donors have been increasingly used due to the scarcity of available grafts. Normothermic ex situ pancreas perfusion (NESPP) can keep grafts metabolically active, potentially allowing for assessment and organ repair, and could improve outcomes of marginal grafts. A novel NESPP technique was developed and tested. Porcine pancreata were removed after a short period of warm ischemia and subjected to 6 h of NESPP. Perfusion parameters, potential graft assessment markers and graft injury were measured. Next, pancreata subjected to 3 h of NESPP were transplanted and animals were followed for up to 3 days. Graft function and injury post-transplantation were evaluated. Using this novel system of perfusion, pancreata were perfused for an extended period of time with minimal edema. Histology at the end of perfusion showed intact islet cells with only mild signs of tissue injury. NESPP transplanted grafts showed immediate function after transplantation, with glucose levels in normal range. NESPP maintains a physiologic environment and excellent graft function without causing significant graft injury. Porcine pancreas transplantation is feasible and allows for in vivo graft assessment of pancreas function and injury after NESPP.

Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function

Ischemia-reperfusion injury (IRI) results in increased rates of delayed graft function and early graft loss. It has recently been reported that hydrogen sulfide (H2 S) protects organ grafts against prolonged IRI. Here, we investigated whether the preservation of pancreas in University of Wisconsin (UW) solution supplemented with AP39, which is a mitochondrial-targeted H2 S donor, protected pancreatic islets against IRI and improved islet function. Porcine pancreata were preserved in the UW solution with AP39 (UW + AP39) or the vehicle (UW) for 18h, followed by islet isolation. The islet yields before and after purification were significantly higher in the UW + AP39 group than in the UW group. The islets isolated from the pancreas preserved in UW + AP39 exhibited significantly decreased levels of reactive oxygen species (ROS) production and a significantly increased mitochondrial membrane potential as compared to the islets isolated from the pancreas preserved in the vehicle. We found that the pancreas preserved in UW + AP39 improved the outcome of islet transplantation in streptozotocin-induced diabetic mice. These results suggest that the preservation of pancreas in UW + AP39 protects the islet grafts against IRI and could thus serve as a novel clinical strategy for improving islet transplantation outcomes.

Pancreas preservation with amphotericin B deteriorates islet yield for porcine islet isolation

Amphotericin B is a crucial agent in the management of serious systemic fungal infections. It is also known to be cytotoxic. In this study, we evaluated the effect of amphotericin B added to the preservation solution on islet yield during islet isolation. Porcine pancreata were preserved in the preservation solution with or without amphotericin B (0.25μg/mL) for approximately 18hours at 4°C, and then islet isolation was performed. An optimized number (1750IE) of isolated islets from each group were transplanted into streptozotocin-induced diabetic mice. The culture of isolated islets and acinar tissue with amphotericin B was also evaluated. The islet yield before and after purification in the amphotericin B (-) group was significantly higher than that in the amphotericin B (+) group. After islet transplantation into diabetic mice, blood glucose levels reached the normoglycemic range, with 50% and 0% of that of the diabetic mice in the amphotericin B (-) and amphotericin B (+) groups, respectively. In the culture study, amphotericin B was found to be cytotoxic to porcine islets and acinar tissue. Amphotericin B added to the preservation solution deteriorates islet yield during porcine islet isolation. Thus, the use of amphotericin B should be considered carefully for the preservation of the pancreas for islet isolation and islet culture before islet transplantation.

Pancreas Preservation in Modified Histidine-lactobionate Solution Is Superior to That in University of Wisconsin Solution for Porcine Islet Isolation.

We previously reported that modified extracellular-type trehalose-containing Kyoto (MK) solution, which contains a trypsin inhibitor (ulinastatin), significantly improved the islet yield compared with University of Wisconsin (UW) preservation, which is the gold standard for organ preservation for islet isolation. In this study, we evaluated the efficiency of a modified histidine-lactobionate (MHL) solution in addition to UW or MK solution. The MHL solution has a high sodium-low potassium composition with low viscosity compared with the UW solution. Moreover, similar to MK solution, MHL solution also contains ulinastatin. Porcine pancreata were preserved in UW, MK, or MHL solution, followed by islet isolation. An optimized number (1500 IE) of isolated islets from each group were then transplanted into streptozotocin-induced diabetic mice. The islet yield before and after purification was significantly higher in the MHL group than in the UW group. On the contrary, the islet yield before and after purification was not significantly different between the MHL and MK groups. Preserving the porcine pancreata in MHL solution improved the outcome of islet transplantation in streptozotocin-induced diabetic mice compared with that in UW solution. Pancreas preservation with MHL solution preserves islet function better than UW solution. The effect of MHL solution is similar to that of MK solution, suggesting that MHL solution can be used as an alternative to MK solution for pancreatic islet transplantation.

Pancreas preservation in extracellular-type p38 inhibitor-containing solution improves islet yield for porcine islet isolation.

For islet transplantation, pancreas preservation and islet isolation activate p38, which is a member of the stress-activated group of mitogen-activated protein kinases (MAPKs). In this study, we evaluated an extracellular-type p38 inhibitor-containing (EP) solution with University of Wisconsin (UW) solution, the gold standard for organ preservation. The EP solution has high sodium-low potassium composition with low viscosity compared to UW solution. Moreover, EP solution contains a recently developed p38 inhibitor (11R-p38I110 ) from our laboratory. Porcine pancreata were preserved in UW, EP, or EP-P solution (EP solution without 11R-p38I110 ), and then islet isolation was performed. An optimized number (1500 IE) of isolated islets from each group were transplanted into streptozotocin-induced diabetic mice. The islet yield before and after purification was significantly higher in the EP group than in the UW group. The islet yield before and after purification was not significantly different between the EP and EP-P groups; however, the EP solution prevented a reduction in the number of islets during culture. Western blot analysis showed that p38 activation was attenuated by EP solution. For islet transplantation into streptozotocin-induced diabetic mice, pancreas preservation in EP solution improved the outcome of islet transplantation. Pancreas preservation with EP solution preserved islet function better than with UW solution. The advantages of EP solution over UW solution may include the inhibition of p38 activity as well as the composition of the solution.

Glucose-Dependent Insulinotropic Polypeptide Is a Pancreatic Polypeptide Secretagogue in Humans.

Glucose-dependent insulinotropic polypeptide (GIP) has been suggested to stimulate the secretion of pancreatic polypeptide (PP), an islet hormone thought to regulate gut motility, appetite, and glycemia. To determine whether human GIP1-42 (hGIP) stimulates PP secretion. As glycemia modulates the secretion of PP, we measured plasma PP concentrations from 2 studies in healthy men (n = 10) and in patients with type 2 diabetes (T2D) (n = 12), where hGIP1-42 had been administered intravenously during fasting glycemia, hyperglycemia (12 mmol/L), and insulin-induced hypoglycemia (targets: 2.5 mmol/L [healthy]; 3.5 mmol/L [T2D]). Porcine GIP1-42 (pGIP) was also infused intra-arterially in isolated porcine pancreata (n = 4). Mean fasting plasma glucose concentrations were approximately 5 mmol/L (healthy) and approximately 8 mmol/L (T2D). At fasting glycemia, PP concentrations were higher during intravenous hGIP1-42 infusion compared with saline in healthy men (mean [standard error of the mean, SEM], net incremental areas under the curves (iAUCs)[0-30min], 403 [116] vs -6 [57] pmol/L × min; P = 0.004) and in patients with T2D (905 [177] vs -96 [86] pmol/L × min; P = 0.009). During hyperglycemic clamping, mean [SEM] PP concentrations were significantly higher during hGIP1-42 infusion compared with saline in patients with T2D (771 [160] vs -183 [117] pmol/L × min; P = 0.001), but not in healthy individuals (-8 [86] vs -57 [53] pmol/L × min; P = 0.69). When plasma glucose levels were declining in response to exogenous insulin, mean [SEM] PP concentrations were higher during hGIP1-42 infusion compared with saline in healthy individuals (294 [88] vs -82 [53] pmol/L × min; P = 0.0025), but not significantly higher in patients with T2D (586 [314] vs -120 [53]; P = 0.070). At target hypoglycemia, PP levels surged in both groups during both hGIP1-42 and saline infusions. In isolated pancreata, pGIP1-42 increased mean [SEM] PP output in the pancreatic venous effluent (baseline vs infusion, 24[5] vs 79 [16] pmol/min x min; P = 0.044). GIP1-42 increases plasma PP secretion in healthy individuals, patients with T2D, and isolated porcine pancreata. Hyperglycemia blunts the stimulatory effect of hGIP1-42 in healthy individuals, but not in patients with T2D.

CD4+ /CD8+ T-cell ratio correlates with the graft fate in pig-to-non-human primate islet xenotransplantation.

Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation. The absolute number and ratio of T-cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells was retrospectively analyzed. The ratio of CD4+ versus CD8+ T cells was significantly reduced due to CD8+ effector memory cells' increase. Correlation analyses revealed that CD4+ /CD8+ , CD4+ /CD8+ naïve, CD4+ naïve/CD8+ naïve, and CD4+ central memory/CD8+ naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4+ /CD8+ cell ratios within the early graft-rejected monkeys (≤60days). This retrospective study demonstrated that CD4+ /CD8+ ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4+ /CD8+ ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation.

Cost and Scalability Analysis of Porcine Islet Isolation for Islet Transplantation: Comparison of Juvenile, Neonatal and Adult Pigs

The limited availability of human islets has led to the examination of porcine islets as a source of clinically suitable tissue for transplantation in patients with diabetes mellitus. Islets from porcine donors are commonly used in both in vitro and in vivo experiments studying diabetes mellitus. However, there are significant differences in quality and quantity of islet yield depending on donor pig age, as well as substantial differences in the costs of pancreas procurement in adult versus neonatal and juvenile pigs. In this study, we compared the total cost per islet of juvenile pig pancreata with that of neonatal and adult pigs. Although adult porcine pancreata yield, on average, more than five times the amount of islets than do juvenile and neonatal pancreata, we found that the high price of adult pigs led to the cost per islet being more than twice that of juvenile and neonatal islets (US $0.09 vs $0.04 and $0.02, respectively). In addition, neonatal and juvenile islets are advantageous in their scalability and retention of viability after culture. Our findings indicate that isolating neonatal and juvenile porcine islets is more cost-effective and scalable than isolating adult porcine islets.

Ex vivo normothermic porcine pancreas: A physiological model for preservation and transplant study

IntroductionAn ex vivo normothermic porcine pancreas perfusion (ENPPP) model was established to investigate effects of machine perfusion pressures on graft preservation. MethodologyNine porcine pancreata were perfused with autologous blood at 50 mmHg (control) pressure. Graft viability was compared against four ex-vivo porcine pancreata perfused at 20 mmHg (‘low’) pressure. Arterio-venous oxygen gas differentials, biochemistry, and graft insulin responses to glucose stimulation were compared. Immunohistochemistry stains compared the cellular viability. ResultsControl pancreata were perfused for a median of 3 h (range 2–4 h) with a mean pressure 50 mmHg and graft flow 141 mL min−1. In comparison, all of the ‘low’ pressure models were perfused for 4 h, with mean perfusion pressure 20 mmHg and graft flow 40 mL.min-1. All pancreata demonstrated cellular viability with evidence of oxygen consumption with preserved endocrine and exocrine function. However, following statistical analysis, the ‘low’ pressure perfusion of porcine pancreata compared favourably in important biochemical and immunohistochemistry cellular profiles; potentially arguing for an improved method for graft preservation. ConclusionENPPP will facilitate whole organ preservation to be studied in further detail and avoids use of expensive live animals. ENPPP is reproducible and mimics a “donation after circulatory death” scenario.

A proteome analysis of pig pancreatic islets and exocrine tissue by liquid chromatography with tandem mass spectrometry

ABSTRACTLiquid chromatography with tandem mass spectrometry (LC-MS/MS) is a proteome analysis method, and the shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolving power. In this study, we analyzed the protein expression in pancreatic tissue by LC-MS/MS. Islets isolated from porcine pancreata (purity ≥95%) and exocrine tissue (purity ≥99%) were used in this study. LC-MS/MS showed that 13 proteins were expressed in pancreatic islets only (Group I), 43 proteins were expressed in both islets and exocrine tissue (Group I&E), and 102 proteins were expressed in exocrine tissue only (Group E). Proteins involved in islet differentiation and cell proliferation were identified in Group I (e.g. CLUS, CMGA, MIF). In addition, various functional proteins (e.g. SCG2, TBA1A) were identified in islet by using the new method of ‘principal component analysis (PCA)’. However, the function of such proteins on islets remains unclear. EPCAM was identified in Group E. Group E was found to include proteins involved in clinical inflammatory diseases such as pancreatitis (e.g. CBPA1, CGL, CYTB, ISK1 and PA21B). Many of these identified proteins were reported less frequently in previous studies, and HS71B, NEC2, PRAF3 and SCG1 were newly detected in Group I while CPNS1, DPEP1, GANAB, GDIB, GGT1, HSPB1, ICTL, VILI, MUTA, NDKB, PTGR1, UCHL3, VAPB and VINC were newly detected in Group E. These results show that comprehensive expression analysis of proteins by LC-MS/MS is useful as a method to investigate new factors constructing cellular component, biological process, and molecular function.

A Method of Porcine Pancreatic Islet Isolation for Microencapsulation.

Since the discovery of insulin by Banting and Best in 1921, the prognosis and treatment options for individuals with diabetes have improved. The development of various insulin types, various oral agents, and insulin pumps have improved the available medical options for individuals afflicted with diabetes. The current need for frequent blood glucose monitoring imposed by multiple daily insulin injections, result in significant life-style challenges for in individuals afflicted with Type 1 diabetes (T1D). In contrast the use of surgical interventions, such as whole organ pancreas transplantation (PT) requires less-intensive glucose monitoring while the organ is viable. Also, isolated human pancreatic islet transplantation (IT) holds similar promise as PT; however, the limited availability of human pancreata exacerbated by, the need for multiple pancreata per individual IT recipient, and issues with prolonged viability, still hamper widespread successful, and routine use of IT. The use of porcine pancreata holds promise as a viable alternative to human pancreas to significantly increase the volume of islets available to meet the needs of millions of patients afflicted with T1D. This chapter outlines our protocol utilized to reliably isolate and microencapsulate porcine islets.

Plasticity and Aggregation of Juvenile Porcine Islets in Modified Culture: Preliminary Observations.

Diabetes is a major health problem worldwide, and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges, and young pigs (3-6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (~3 months; 25 kg Yorkshire pigs) immediately following procurement or after 24 h of hypothermic machine perfusion (HMP) preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology, and in vivo function. Islet yields were highest from HMP-preserved pancreata (2,242 ± 449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 μm), and purity was on average 63 ± 6%. Morphologically, islets appeared as clusters on day 0, loosely disaggregated structures at day 1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day 6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki-67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 175 ± 16 nmol/min/mg DNA, and physiological function was intact by glucose stimulation after 6-8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) of transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation require specialized conditions. These immature islets undergo plasticity in culture and form fully functional multicellular structures. Further development of this method for culturing immature porcine islets is expected to generate small pancreatic tissue-derived organoids termed "pancreatites," as a therapeutic product from juvenile pigs for xenotransplantation and diabetes research.

Current status and future of clinical islet xenotransplantation.

β-Cell replacement therapy, including allogeneic pancreas and islet transplantation, can normalize HbA1c levels in unstable type 1 diabetic (T1D) patients, but a donor shortage is a serious issue. To overcome this problem, xenotransplantation is an attractive option. In fact, islet transplantation from porcine pancreata was performed in the 1990s, which opened the door for islet xenotransplantation, but the possibility of porcine endogenous retrovirus (PERV) infection was raised, which has restricted progress in this field. The International Xenotransplantation Association published a consensus statement on conditions for undertaking clinical trials of porcine islet products in T1D to restart islet xenotransplantation safely. Clinical porcine islet xenotransplantation was restarted under comprehensive regulations in New Zealand. In addition, newly emerged gene-editing technologies have activated the xenotransplantation field. Islet xenotransplantation is becoming a clinical reality, with the results of recent studies showing promise to advance this field.

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.

51. Humidified oxygen gas perfusion (persufflation) in organ preservation and cryobanking

It is widely recognized that exposure to ischemia and its metabolic and sequelae during procurement and preservation results in tissue injury and/or death and that normothermic reperfusion during transplantation further amplifies the damage. This is also now a recognized issue in organ cryopreservation with data suggesting that inadequately preserved (damaged) organs may not be successfully cryopreserved and rewarmed for transplantation. Published literature reports demonstrate that enhanced oxygenation during organ preservation for kidney, heart, pancreas, and liver, results in improved organ viability, leading to better short and long-term outcomes including limiting chronic rejection in a porcine kidney and heart transplant model. Persufflation (PSF) the flow of hypothermic humidified oxygen gas through the vasculature of organs has been shown to be effective in oxygenating and preserving as well as reconditioning a variety of organs including kidneys, livers, hearts, and pancreata from expanded criteria donors (including Donors after Cardiac Death) in a variety of small and large animal models with excellent outcomes. Persufflation is currently in a clinical trial for reconditioning (after hypothermic static storage) and transplanting livers that would have otherwise been discarded in Europe. PSF also extended significantly the allowable window for organ preservation (for example 14 h instead of 4–6 for the heart and 24 h instead of 8–12 h for the pancreas) without compromising outcomes. However, perhaps due to the unavailability of a “turnkey” portable persufflator and the requirement for use of pressurized oxygen gas cylinders, PSF has not been widely used clinically and has been until recently limited to reconditioning organs after prolonged cold ischemia times (shipped on hypothermic static storage). The development and current availability of a novel portable battery operated electrochemical oxygen generator (“persufflator”) that can be used during flight and deliver humidified oxygen gas to the vasculature of organs may change that. This PSF has been extensively tested with porcine and human pancreata and to a limited extent with porcine kidneys, livers, hearts and composite tissues. It is currently in pilot clinical testing in Canada for pancreas preservation prior to rewarming for digestion and islet purification for clinical islet allo transplantation. In this paper we present we discuss the utility of PSF in organ preservation and in particular organ (re) conditioning prior to cryopreservation as well as during thawing and rewarming prior to transplantation. Financial Support: NIH/NIDDK SBIR R44DK070400.

Temperature profiles of different cooling methods in porcine pancreas procurement.

Porcine islet xenotransplantation is a promising alternative to human islet allotransplantation. Porcine pancreas cooling needs to be optimized to reduce the warm ischemia time (WIT) following donation after cardiac death, which is associated with poorer islet isolation outcomes. This study examines the effect of four different cooling Methods on core porcine pancreas temperature (n = 24) and histopathology (n = 16). All Methods involved surface cooling with crushed ice and chilled irrigation. Method A, which is the standard for porcine pancreas procurement, used only surface cooling. Method B involved an intravascular flush with cold solution through the pancreas arterial system. Method C involved an intraductal infusion with cold solution through the major pancreatic duct, and Method D combined all three cooling Methods. Surface cooling alone (Method A) gradually decreased core pancreas temperature to <10 °C after 30 min. Using an intravascular flush (Method B) improved cooling during the entire duration of procurement, but incorporating an intraductal infusion (Method C) rapidly reduced core temperature 15-20 °C within the first 2 min of cooling. Combining all methods (Method D) was the most effective at rapidly reducing temperature and providing sustained cooling throughout the duration of procurement, although the recorded WIT was not different between Methods (P = 0.36). Histological scores were different between the cooling Methods (P = 0.02) and the worst with Method A. There were differences in histological scores between Methods A and C (P = 0.02) and Methods A and D (P = 0.02), but not between Methods C and D (P = 0.95), which may highlight the importance of early cooling using an intraductal infusion. In conclusion, surface cooling alone cannot rapidly cool large (porcine or human) pancreata. Additional cooling with an intravascular flush and intraductal infusion results in improved core porcine pancreas temperature profiles during procurement and histopathology scores. These data may also have implications on human pancreas procurement as use of an intraductal infusion is not common practice.

Islet Purification Method Using Large Bottles Effectively Achieves High Islet Yield from Pig Pancreas

Porcine islets are a promising resource for xenotransplantation. However, low efficacy of islet isolation because of their marked fragility remains a problem. Recently we found that the standard purification method using COBE 2991 cell processor (COBE) with Ficoll density gradient solution damaged islets mechanically by high shearing force. In this study, we evaluated our new purification method using large plastic bottles for the efficacy of islet purification. Ten porcine pancreata were used. The average warm ischemic time was over 40 min; therefore, these pancreata were considered to be in a marginal condition. After digestion, the digested tissue was divided into three groups. Each group was purified using either top loading method with bottle (top group) or bottom loading method with bottle (bottom group) or standard COBE method (COBE group). Islet yield per pancreas weight (IEQ/g) and the rate of postpurification recovery in the top group were significantly higher than the COBE group (top: 8060 ± 1652 IEQ/g, bottom: 4572 ± 614 IE/g, COBE: 3900 ± 734 IE/g. p < 0.02 in top vs. COBE; top percentage of recovery: 99.3 ± 12.3%, bottom: 62.6 ± 8.8%, COBE: 49.5 ± 6.7%, p < 0.02 in top vs. bottom and COBE). The average sizes of purified islets in the top and bottom groups were significantly larger than COBE group (Average diameter top: 156 ± 8 μm, bottom: 147 ± 6 μm, COBE: 119 ± 6 μm, p < 0.01 in top vs. COBE and in bottom vs. COBE), which indicated that bottle method can reduce shear force during purification. Our new purification using top loading bottle method enabled us to obtain a high yield of porcine islets from marginal pancreata.